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UBC Theses and Dissertations

Characterization of gene expression patterns in the developing natural killer cell Birro, Natasha Diane 2002

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C H A R A C T E R I Z A T I O N O F G E N E E X P R E S S I O N P A T T E R N S I N T H E D E V E L O P I N G N A T U R A L K I L L E R C E L L by Natasha Diane Birro B.Sc., The University of Western Ontario 1999 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Pathology; Genetics Programme) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A M a y 2002 © Natasha Diane Birro , 2002 In p resen t ing this thesis in partial fu l f i lment of t h e requ i rements fo r an advanced degree at the Univers i ty o f Brit ish C o l u m b i a , I agree that t h e Library shall make it f reely available f o r re ference and study. I fu r ther agree that permiss ion f o r extens ive c o p y i n g o f th is thesis f o r scholar ly pu rposes may b e g ran ted by the head of my d e p a r t m e n t or by his o r her representat ives. It is u n d e r s t o o d that c o p y i n g or pub l i ca t i on o f this thesis f o r f inancial gain shall n o t b e a l l o w e d w i t h o u t m y w r i t t e n permiss ion . The Univers i ty o f Brit ish C o l u m b i a Vancouver , Canada D E - 6 (2 /88) Abstract Relatively little is known about development of the Natural Ki l l e r (NK) cell. Studies in the past have focused on characterizing the phenotype of the N K cell, and evaluating its development by manipulating hematopoietic stem cells in vitro. When I began my study, an in vitro culture system had been recently developed in my laboratory in which embryonic stem cells are induced with various cytokines and growth factors to differentiate into N K cells. This system provides a medium where N K cell development can be researched from an earlier stage than previously possible. The objective of my work was to use this culture system to investigate the expression patterns of five genes which are known to play a role in lymphoid cell development based on gene knock-out studies. Id-2 is an inhibitor of D N A binding protein, Ikaros-1 and Ets-1 are transcription factors, Notch-1 is a transmembrane receptor and Jagged-1 is a ligand for Notch. Using R T - P C R and phospho-image analysis, the expression patterns of these genes were assessed from various numbers of cells, and at five different points along the developmental pathway of the N K cell. Id-2 seemed to be expressed consistently through out the system. Ikaros-1 and Ets-1 were interesting in that they showed an on/off expression pattern, Ets-1 with little expression on days 8 and 14, and Ikaros-1 with no expression on days 8 and 14. This implies that perhaps these transcription factors are not needed at the initial stage of N K cell differentiation from a common lymphoid precursor. Notch-1 and Jagged-1 were particularly interesting in that their expression patterns did not parallel each other. These results suggest that Jagged-1 may not be the ligand of choice for proper N K cell development as Notch levels were lower than expected. These iii studies have begun to give us a better understanding of gene expression in our system, and provide clues as to what role certain gene may play. I V Table of Contents Abstract ii Table of Contents iv List of Figures viii List of Abbreviations ix Acknowledgements xi Chapter 1 Introduction 1 1.1 Natural Killer Cell Characteristics 1 1.1 (a) Physical Characteristics 1 1.1 (b) Role in Infection 2 1.1 (c) Anti-Tumour Role 3 1.2 Mode of Action 4 1.2 (a) The Major Histocompatibility Complex-class 1 4 1.2 (b) Missing Self Hypothesis 4 1.3 MHC-1 Specific Receptors In Mice 5 1.3 (a) M H C - 1 Specific Receptors 5 1.3 (b) Ly49 family of receptors 6 1.3 (c) The C D 9 4 / N K G 2 family of receptors 6 1.3 (d) Inhibition Mechanism 7 1.3 (e) Activation Mechanism 8 1.3 (f) Receptor Acquisition 9 1.4 Development 10 V 1.4 (a) N K cell development 10 1.4 (b) In Vitro N K cell development 12 1.5 Genes Involved in N K cell Development 14 1.5 (a) Ikaros 14 1.5 (b) Ets 17 1.5 (c) Id-2 19 1.5 (d) Notch-1 and Jagged-1 21 1.6 Thesis Objectives 23 Chapter 2 Materials and Methods 25 2.1 Oligonucleotides 25 2.1 (a) Oligonucleotide design 25 2.1 (b) Oligonucleotide specificity 25 2.2 Control Tissues 25 2.2 (a) Spleen 25 2.2 (b) Thymus 27 2.2 (c) Bone Marrow 27 2.3 Cell Culture 28 2.3 (a) Culture System 28 2.3 (b) Staining and Sorting 29 2.3 (c) Culture of L A K cells 29 2.4 R T - P C R 30 2.4 (a) R T - P C R for Controls 30 vi 2.4 (b) R T - P C R For a Limiting Number of Hematopoietic Cells 31 2.4 (c) R T - P R C for 5 Genes of Interest 34 2.5 3 2 P Labelling of Oligonucleotides 35 2.6 Hybridization 35 2.6 (a) Controls 35 2.6 (b) Hybridization of Oligonucleotides Specific For Genes of Interest 36 2.7 Exposure to Phosphoimager 36 Chapter 3 Results 38 3.1 Oligonucleotide design 39 3.2 G A P D H 41 3.3 Id-2 41 3.4 Jagged-1 and Notch-1 45 3.5 Ikaros-1 51 3.6 Ets-1 54 3.7 OP-9 Gene Expression 57 Chapter 4 Discussion 59 4.1 In Vitro Generation of NK-like cells 59 4.2 Ikaros-1 60 4.3 Ets-1 62 4.4 Id-2 65 4.5 Jagged-1 and Notch-1 66 vii 4.6 Stromal Environment 69 4.7 Conclusion 69 Bibliography 71 Appendix A 79 V l l l List of Figures Figure 2.1 P R C primers 26 Figure 2.2 Oligonucleotide probes 26 Figure 2.3 P C R control conditions 32 Figure 2.4 P C R sample conditions 32 Figure 2.5 Oligonucleotide melting temperature equation 32 Figure 2.6 Five time points chosen for gene expression analysis along and N K cell developmental pathway 33 Figure 3.1 Controls 40 Figure 3.2 G A P D H 42 Figure 3.3 Id-2 43 Figure 3.4 Jagged-1 46 Figure 3.5 Notch-1 49 Figure 3.6 Ikaros-1 52 Figure 3.7 Ets-1 55 Figure 3.8 OP-9 cells 58 Figure 4.1 Five time points chosen for analysis with results 61 ix List of Abbreviations A D C C antibody dependent cell-mediated cytotoxicity B 6 C 5 7 B L / 6 b H L H basic helix-loop-helix transcription factor B S A bovine serum albumin C B F C C A A T binding factor c D N A complementary deoxyribonucleic acid d d H 2 0 distilled water D A P - 1 2 D N A X adaptor protein-12 D E P C - H 2 0 Diethyl pyrocarbonate treated water D M E M Dulbecco's Modif ied Eagle's Medium D N A deoxyribonucleic acid d N T P deoxyribonucleotide triphosphate E D T A ethylenediamine tetra acetate E B S Ets binding site E B V Epstein-Barr virus E G F epidermal growth factor E S cell embryonic stem cell E t O H ethanol F A C S fluorescence activated cell sorting F B S fetal bovine serum F1TC fluorescen isothiocyanate GIT guanidine isothiocyanate G A P D H glyceraldehydes 3-phosphate dehydrogenase H2 histocompatibility complex 2 I F N interferon Ig immunoglobulin IL interleukin I M D M Iscove's Modif ied Dulbecco's Medium IT A M immunoreceptor tyrosine-based activation motif I T I M immunoreceptor tyrosine-based inhibition motif L A K lymphokine activated killer L F A - 1 lymphocyte function-associated antigen-1 m A b monoclonal antibody M H C major histocompatibility complex M i - 2 - H D A C M i - 2 histone deacetylase m S i n 3 - H D A C mSin3 histone deacetylase M T G monothioglycerol N K cell natural killer cell P C R polymerase chain reaction P E phycoetythrin PI propidium iodide P T K protein tyrosine kinase R P M rotation per minute R T - P C R reverse transcriptase-polymerase chain reaction SDS sodium dodecyl sulphate S C F stem cell factor SH2 Src-homology 2 S H P SH2-domain-containing phosphatase X SSC sodium chloride-sodium citrate buffer SWI/SNF switch/sucrose non-fermentor chromatin remodelling molecule TAP transporters associated with antigen processing TdT terminal transferase Acknowledgements A special thanks to: my supervisor Fumio Takei whose patience is infinite, and who was always available to me to guide, correct and encourage my work. To Dixie Mager my co-supervisor, who has contributed a great deal to my progress through constructive feedback at those early morning lab meetings. Thanks to the members of my lab: Reza Marwal i , Lixen Zhou, Nooshin Tabatabaei, Lisa Dreolini, Linnea Veinotte, and Ashleen Shadeo. I want to extend a special acknowledgement to two lab members in particular. To Motoi Maeda who has spent a great deal of time and energy in a purely altruistic fashion, generating the culture system that was such an essential part of my thesis. Thank you for your incredible patience, and for your willingness to teach me everything I was so inclined to question. Y o u are a great teacher and an incredibly competent scientist. To Rebecca Lian. Thank you not only for the enormous amount of work you put into the culture system solely for my use, but for your willingness to listen to my questions and my complaints. Thanks for being more than a mentor. To my Vancouver buddies: Jennifer Hobday, Robert Artibese, Layne Harvey, Kate Harvey, Michael St Pierre, Christina Trotter, Grant Lamount, and Aver i l Sheppard. Y o u all have changed my life forever. Vancouver was carved into my heart thanks to your friendships. The laughs, the hikes, the climbing, the camping and let's not forget the Ultimate Frisbee. Thanks for all the things that brought happiness to me over the past three years. To my Toronto buddies: Thyrza Naraine, Jaya Ramlakhan, Karen Paul, Tamara Ferguson, Lisa Wust, Anne Molnar, Al l i son Stott. Y o u are more than friends, you are family. Y o u hold my past and w i l l continue to influence my future. To my family: Dad (Mustafa Birro), M o m (Sarah Birro) and my brother A m i r Birro. Y o u are my foundation. Thank you for your strength, support, and unconditional love. Thanks for your encouragement went things were hard, and for you persistent belief that I can do what ever I set my heart out to do. Lastly, to my husband... my best friend Jason McNamee. Your guidance, advice and encouragement through the past 3 years have been invaluable. With your strength and love, I can complete any task and begin anew with complete confidence. Besides being a diligent scientist, a compassionate friend, and a pillar of strength, you are my inspiration. 1 Chapter 1 Introduction 1.1 Natural Ki l l e r Cel l Characteristics 1.1 (a) Physical Characteristics Natural Ki l l e r cells ( N K cells) were first discovered because of the ability they possess to k i l l tumour cells in vitro, without prior sensitization. This process was termed natural ki l l ing. ( K i m et al. 2000). They are bone marrow derived lymphocytes that share a common progenitor with T cells. They account for 5-10% of human peripheral blood leucocytes and they can k i l l a wide range of targets. In fact, ki l l ing by N K cells can be detected within 24 hours of a viral infection, and this attack may persist until a T cell response mounts. During this attack N K cells produce a wide range of factors, such as interferon y, tumour necrosis factor a , and granulocyte/macrophage colony-stimulating factor, which provoke an immune response (O'Callaghan 2000). N K cells are distinguished from other lymphocytes by the absence of antigen receptor C D 3 and by the presence of CD56 (NK1.1 or D X 5 in mouse). The majority also express C D 16. This latter receptor enables these cells to mediate antibody-dependent cell-mediated cytotoxicity ( A D C C ) via its low-affinity for the Fc region of IgG (Lanier 2000). The roles N K cells play in both the innate and adaptive immune system makes them unique and effective immune cells. N K cells mediate cytotoxicity through release of granules containing proteins such as perforin and granzymes. Perforin monomers incorporate into the cell membrane, and via a calcium dependent mechanism, polymerise to form pores in the membrane (Young and Cohn 1987). Granzymes, which are serine proteases, cause apoptosis via rapidly inducing degradation and fragmentation of D N A (Heusel et al. 1994). The importance of N K cells was revealed when a rare human disorder, which involved a complete absence of N K cells, was characterized. Patients which present this disorder have difficulty controlling viral infections(Lanier 2000). A s well as their involvement in controlling viral infections, N K cells play an important role in suppressing tumour metastasis and outgrowth in mice (K im et al. 2000). 1.1 (b) Role in Infection N K cells recognize virally infected cells by two ways. Firstly, they may recognize antibody-coated cells. Cells that have been virally infected wi l l present viral antigens on their surface, and this w i l l attract antibodies. Specifically, CD16 (an Fc receptor on N K cells) recognizes I g G l and IgG3, and this triggers a cytotoxic attack of the virally infected cell (via A D C C ) (Lanier et al. 1988). Secondly, virally infected cells may down-regulate class I major histocompatibility complex ( M H C ) . Through a mechanism described as the "missing-self hypothesis", ki l l ing of virally infected cells w i l l ensue (Mil ler 2001). Virus infected cells release Interferon-oc/p (IFN-a/|3) which enhances N K cell cytotoxicity. A s well , virally infected cells can release IL-12, which causes the N K cell to release Interferon-y, and IFN-y can activate important pathways associated with antiviral functions (Biron and Brossay 2001). 1.1 (c) Anti-tumour Role Natural Ki l l e r cells represent a group of lymphocytes which possess spontaneous cytolytic activity against some tumour cells. They are presumed to play a role in the response against tumours in vivo as they can eliminate circulating tumour cells (Chong et al. 1998). Animal models have been a source of great information regarding the role of N K cells in anti-tumour activity. One study showed an increase in long-term survival and a 40% complete tumour regression in hepatic metastatic breast cancer in a murine model, after adenoviral transduction of IL-12. Antibody depletion of N K cells but not C D 4 or C D 8 T-cells abrogated this effect. The IL-12 was presumed to mediate the effect by increasing both the cytotoxicity of N K cells and their IFN-y production (Miller 2001). Another study produced a transgenic mouse with a deficiency in N K 1 . 1 + CD3" cells. This study allowed N K cell function to be ascertained in vivo. The transgenic mice had no in vitro natural ki l l ing activity. Using radiolabeled Y A C - 1 cells (a widely accepted target of N K cells), it was demonstrated that wild-type mice were able to eliminate tumour cells in vivo, whereas transgenic mice showed >70 fold higher levels of radioactivity indicating lack of cell death from N K cells (Kim et al. 2000). L o w expression of M H C class I by tumours such as B16 melanoma leads to poor recognition by T cells, however favours N K cell activation. N K cell activating receptors, while less understood, also are assumed to play some anti-tumour roles (Chong et al. 1998). 4 1.2 Mode of Act ion 1.2 (a) The Major Histocompatibility Complex-class I The major histocompatibility complex ( M H C ) class I is a family of cell-surface glycoproteins that consists of a heavy chain and P2 microglobulin chain that are non-covalently bound to each other. Its primary role is to present endogenous antigens to elicit a cytotoxic response from CD8+ T lymphocytes (Trowsdale 1993). It also plays a role in N K cell mediated cell lysis. The M H C is the most polymorphic region of the human genome. The murine class la molecules consist of H - 2 K , H-2D, and H - 2 L ( H L A -A , H L A - B , and H L A - C in humans) (O'Callaghan 2.000). There are also three non-classical M H C molecules, Q a - l b being one of importance in mice. This protein binds to the highly conserved hydrophobic leader peptide of the classical mouse M H C I molecules. Hence, the classical M H C I molecules must be present in cells for the stable mature non-classical M H C molecule Q a - l b to form and migrate to the cell surface(0'Callaghan 2000). 1.2 (b) Missing Self Hypothesis Based on research that showed N K cells preferentially killed mouse tumour cells that lacked self- M H C class I molecules, it was proposed that a mechanism exists to eliminate cells with aberrant M H C expression (Ljunggren and Karre 1990). Recognition of M H C by human N K cells was shown by the observation that human N K cells lysed M H C class I deficient EBV-transformed B lymphoblastoid cell lines (Lanier 1998). Ultimately there 5 is an inverse relationship between target cell susceptibility to lysis by N K cells and target cell expression of M H C class I proteins- (Vales-Gomez et al. 2000). According to this "missing self hypothesis", one function of N K cells then, is to recognize and terminate cells that lack the self M H C I molecules (Ljunggren, Karre 1990). 1.3 M H C - 1 Specific Receptors In Mice 1.3 (a) M H C - 1 Specific Receptors The cytotoxicity of N K cells is regulated by a balance between signals received from activating and inhibitory cell surface receptors. The interaction of receptors with ligands on potential target cells results in both inhibitory and activating signals and the balance of these signals determines the behaviour of the N K cell (Vales-Gomez et al. 2000). N K cell receptors fall into two types: the C-type ( C a 2 + dependent) lectin superfamily, which are disulfide-linked dimeric type II integral membrane proteins; and the immunoglobulin-like receptors, which are type I integral membrane proteins (Yokoyama 1998). Murine cytotoxicity is regulated by two families of receptors both belonging to the C-type lectin superfamily. The Ly49 family is specific for classical M H C I, and the C D 9 4 / N K G 2 family is specific for the non-classical M H C I (Qa- l b ) (Williams et al. 2000). Q a - l b is expressed on the cell surface after binding signal peptides from classical M H C I in a T A P dependent manner (Lanier 2000). Activating receptors remain poorly characterized. Inhibitory receptors specific for self - M H C I w i l l interact with it, and as a result receive an inhibitory signal which prevents an attack on the potential target. Inhibitory receptors mediate this effect through a sequence in the cytoplasmic domain termed immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Yokoyama 1998). 1.3 (b) Ly49 family of receptors M H C class I recognizing N K receptors were first identified in mouse as the Ly49 proteins, which are homodimeric type II lectins. Humans do not use Ly49, although a non-functional Ly49 receptor has been found in man (Westgaard et al. 1998). The Ly49 family consists of at least 14 closely related genes, ten of which are expressed in B6 mice (Ly49 A-J ) . A l l genes are located in the Natural Ki l le r gene complex on the mouse chromosome 6 (Yokoyama 1998). A diverse repertoire of Ly49 receptors is the result of different Ly49 genes being expressed in overlapping subsets within the N K cell population (Lanier 1998). Engagement of specific M H C I molecules by Ly49 members which posses an intracellular I T I M motif has been shown to be the inhibiting mechanism that is capable of overriding positive activating signals. O f the Ly49 family, Ly49D and L y 4 9 H are activating receptors. They lack an I T I M motif (Mason et al. 1997) but have a charged amino acid in their transmembrane domain that mediates association with D A P - 1 2 , a signalling molecule having an I T A M (immunoreceptor tyrosine-based activation motif)(Lanier 2001). 1.3 (c) The C D 9 4 / N K G 2 family of receptors Human, rat and mice express inhibitory receptors for non-classical M H C I molecules ( Q a - l b in mice, H L A - E in humans). The receptor is a heterodimeric, disulfide bonded 7 molecule composed of a CD94 and a N K G 2 subunit (Lanier 2000). The genes for these molecules, like those for Ly49s, are located in the N K gene complex (Yokoyama 1998). The signal transduction capacity of the receptor stems from the cytoplasmic domain of the N K G 2 subunit (Yokoyama 1998). Although N K G 2 A and B appear to be alternatively spliced from the same gene, N K G 2 C - F are the products of distinct genes. N K G 2 F has been found only in humans. A distinction lies within the family in that N K G 2 A , B and F contain an I T I M in their cytoplasmic portion and thereby transmit inhibitory signals where as N K G 2 C , D , and E lack cytoplasmic ITIMs. N K G 2 C and E are associated with D A P - 1 2 and N K G 2 D is associated with DAP-10 . These molecules facilitate the transmission of an activation signal. It has been shown that C D 9 4 / N K G 2 participates in the protection of self cells from N K cell cytotoxicity through Qa-1 recognition independently of the Ly49 family (Toyama-Sorimachi et al. 2001). 1.3 (d) Inhibition Mechanism Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are located in the cytoplasmic domains of Ly49 inhibitory receptors. While CD94 has only a short cytoplasmic tail, N K G 2 A contains two cytoplasmic ITIMs (Carretero et al. 1998). When Ly49 or C D 9 4 / N K G 2 receptors on N K cells bind their M H C I ligand on target cells, the I T I M residues become tyrosine-phosphorylated by Src-family protein tyrosine kinases (PTK) . SHP-1 is then recruited to the phosphorylated ITIMs, and this may result in eventual co-aggregation of SHP-1 with activating receptors. This inhibits P T K dependent N K cell activation (Lanier 2000) (Leibson 1997). It is also thought recruitment of SHP-1 8 and SHP-2, results in the dephosphorylation of substrates critical for cellular activation (McVicar and Burshtyn 2001). 1.3 (e) Activation Mechanism A D C C mediated ki l l ing in mice, as mentioned in 1.1(b), is initiated when a multimeric receptor complex consisting of C D 16 associated with a homodimer y subunit, engages in low affinity binding to the Fc portion of antibodies bound to cell associated antigens. The IT A M motif in the y subunit of the receptor complex is phosphorylated and in murine N K cells, this y subunit couples to the protein kinase Syk. This activation of P T K s modifies multiple downstream signalling events that result in the development of A D C C and cytokine release (Leibson 1997). It is thought that there is no single triggering receptor for natural cytotoxicity. On the contrary, it is believed there are a variety of activating cellular receptors. One thought is that adhesion molecules such as L F A - 1 act with other cell surface molecules to trigger N K cell activation. Isoforms of inhibitory receptors (Ly-49, and C D 9 4 / N K G 2 ) exist on N K cells, and differ from their inhibitory counterparts in that they do not possess an I T I M (Leibson 1997). The mouse Ly49 D receptor has been shown to activate N K cell cytotoxicity. It interacts with the M H C I antigen H-2Dd, but not with H-2Db or H-2Dk. (Nakamura et al. 1999). It also associates with D A P 12. Thus, lysis of class I M H C -bearing targets by N K cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly49 receptors (Nakamura et al. 1999). 9 The N K G 2 D receptor has received attention for its role in activating N K cells. It is only distantly related to the other N K G 2 isoforms, and forms a homodimer as opposed to pairing with CD94. Recent evidence suggests that it associates with signalling subunit D A P 10 and binds either of two ligands, H-60, or Rae 1(3. These two ligands are not expressed by normal cells, but are up-regulated in certain tumour cells. This evidence supports the notion that N K G 2 D is a stimulatory receptor that N K cells employ to attack tumour cells (Diefenbach et al. 2001) (Yokoyama 2000). 1.3 (f) Receptor Acquisition Natural Ki l l e r cells in mice express at least one inhibitory receptor and most N K cells express one or more members of the Ly49 family (Dorfman and Raulet 1998). Ly49 receptors are expressed on overlapping subsets of N K cells. This means a single N K cell may express several receptors. Most of the Ly49 receptors bind one or more of the M H C I molecules tested, and show specificity for different class I alleles (Roth et al. 2000). This ideally generates diversity in the N K cell repertoire, which allows for targeting of a broad scope of M H C I deficient cells. The overall pattern of expression indicates that a stochastic mechanism governs which Ly49 receptors an N K cell w i l l express. However, studies have revealed that this process is not stochastic in its entirety since frequencies of N K cells expressing different Ly49 receptors are influenced by host M H C I expression (Dorfman, Raulet 1998). While studies have provided evidence to support the notion that receptor acquisition is not entirely random, single cell R T - P C R has shown that there is a highly diverse pattern of coexpression of the different receptor genes in each adult N K cell. O f 62 adult N K 10 cells examined, 42 different combinations of receptor coexpression was observed. This same study showed neonatal N K cells express mostly N K G 2 A , but not Ly49s (Kubota et al. 1999). It suggests that expression of N K G 2 A precedes Ly49 expression. 1.4 Development 1.4 (a) N K cell development N K cells develop naturally in the bone-marrow, and they mature predominantly in extrathymic locations. Data exists to substantiate the idea that N K cells derive from proliferating bone marrow precursors, such as the fact that an intact marrow environment is necessary for N K cell development (Carson and Caligiuri 1996). Although N K cell progenitors have been discovered in the fetal thymus, it is important to note that this thymus environment is not necessary for development since hematopoietic stem cells in bone-marrow generate N K cells in athymic mice. It is thought that cells from the bone marrow expressing 11-15 receptor, 11-2 receptor beta chain, and are CD122+, are in fact N K cell precursors and represent the earliest adult B M precursor uniquely restricted to the N K cell lineage (Rosmaraki et al. 2001). In addition to N K cell precursors in the bone-marrow, a population of T / N K committed progenitors has been identified. They are phenotypically different from multipotent precursor thymocytes. It was shown that exposure of these cells to a thymus micro-environment results in commitment to the T-cell lineage, whereas exposure to bone-marrow microenvironment results in mature N K cell generation (Carlyle et al. 1997). More recent studies have shown that the T / N K bipotent cell does not migrate to 11 the bone-marrow, but is responsible for producing N K cells solely in the thymus. The earliest hematopoietic cells to colonize the thymus are thought to be multipotent lymphoid-committed precursors. After exposure of these cells to the thymic micro-environment, the precursors lose B lymphoid potential and are considered T / N K cell bipotent (Carlyle et al. 1998). N K cells, although not dependent on thymus for development, can develop in the thymus, and seem to stem from a common thymic progenitor with T lymphocytes (Carlyle et al. 1998). The earliest that N K cells are seen in the thymus are at day 13 of gestation. Here they stem solely from these thymic T / N K cell precursors (Carlyle et al. 1998). The above discussion illustrates two lines of thought on N K cell precursors. On the one hand it is argued that precursors exist in the bone-marrow. On the other hand, it is argued that precursors are a bipotent T / N K cell that is derived in the thymus. Both arguments may be valid. Together, the above findings suggest that fetal N K cell differentiation occurs from T / N K cell precursors, and may be restricted to the thymus until peripheral sites of N K lymphopoiesis can be established. This helps to explain why there is a low level of N K cells in the neonatal circulation. The N K cells produced here may not reach the periphery. The bone-marrow may be primarily involved in peripheral N K cell production. Whether the progenitors to N K cells in the bone-marrow are the T / N K cell progenitors that have migrated from the thymus remains unknown. It is unlikely as N K cell development occurs in athymic mice, and multipotent lymphoid-committed precursors differentiate into T / N K cells in the thymus (Carlyle et al. 1998). 12 1.4 (b) In Vitro N K cell development There have been systems established to induce the differentiation of N K cells from multipotent bone-marrow progenitor cells, and from embryonic stem cells. The rationale is to generate a system to develop N K cells in vitro, and then to manipulate the system experimentally to study N K cell development further. Previous studies have established that an OP9 stromal layer not only supports N K development in vitro, but supports acquisition of Ly49 molecules in the developing N K cell (Williams et al. 1999). Stromal cells participate in supporting the proliferation and survival of N K cells. Importantly, a stromal cell line provides the signals necessary for both initiation of Ly49 expression and efficient clonal growth (Roth et al. 2000). In vitro systems utilizing bone marrow precursor cells are able to generate Ly49 expressing N K cells. Precursors are cultured in the presence of cytokines and a bone marrow stromal layer. It was found that Ly49 expression required signals from stromal cells. A s well , some studies have found M H C class I expression has had a significant effect on determining the sequential expression of inhibitory Ly49 receptros (Roth et al. 2000). A clonal assay was developed whereby bone marrow progenitors were isolated and cultured in 11-7, stem cell factor (SCF) and flt3 ligand for 5 days to generate N K precursor cells. These cells were then cultured for 6-7 days in 11-2 to produce NK1.1+, L y 4 9 + N K cells. A significant body of evidence suggests that 11-15 is the physiological cytokine responsible for N K development in vivo (Lodolce et al. 1998). In vitro high doses of 11-2 can substitute for 11-15. N K cells generated from this culture system are 13 L y 4 9 + only in the presence of a confluent monolayer of OP9 stroma (Williams et al. 2000). While in vitro systems utilizing hematopoietic progentors are a useful method of gaining insight into N K cell biology, there are numerous advantages to using an embryonic stem cell differentiation system for characterizing genes involved in lymphoid development. ES cells can be genetically manipulated and studied in vitro, without the worry of embryonic lethality. ES cells are totipotent cells derived from the inner cell mass of a blastocyst. In vivo they retain the ability to contribute to all cell lineages. In vitro, they can differentiate into complex structures called embroyoid bodies, which contain a number of different cell types. There is much potential in studies that focus on embryonic stem cells as a means of understanding the function of genes in development. In vitro manipulation of the ES cells provides insights into what genes may be involved and to what degree (Keller 1995). Many studies have utilized an in vitro culture system of ES cells to understand hematopoietic cell development. Murine ES cells can be maintained in a totipotent stage indefinitely. A t any time researchers can differentiate these cells in vitro into hematopoietic precursors of almost all the colony forming cells in bone marrow (Snodgrass et al. 1992). Initially studies looked at correlations of cytokine gene expression, their receptors, and cell surface markers. Results showed definite order to expression of genes during development (Schmitt et al. 1991). ES cell culture systems are particularity important when investigating gene expressision which preceeds hematopoietic development. ES cell cultures allow us to begin investigation at an earlier stage and to allows us to watch changes in development through a broader scope. To 14 date relatively little is known about how C D 9 4 / N K G 2 receptors are aquired during development. Most fetal N K cells express C D 9 4 / N K G 2 but not Ly49 receptors. In one study, it was an ES cell culture system which allowed for investigation of the acquisition of C D 9 4 / N K G 2 receptors in the N K cell developmental pathway (Lian et al. 2002). 1.5 Genes Involved in N K cell Development There are many genes that potentially play a role in N K cell development. The IL-15 receptor gene is critical for N K cell maturation (Lodolce et al. 1998). It supports development of C D 3 4 + stem cells into N K cells in vitro and mediates induction of C D 9 4 / N K G 2 inhibitory receptors in early N K precursors (Lian unpublished). Interferon regulatory factors (IRFs) are a family of nine transcriptional regulators. They are thought to play a role in N K cell development as knock-out studies result in a marked reduction in N K cell number (Lian unpublished). T C F - 1 , a trans-acting factor, is necessary for regulating the expression of the Ly49 inhibitory receptors in maturing N K cells (Held et al. 1999; Toor et al. 2001). These are just a few examples of genes that may influence N K cell development. There are however five genes of particular interest concerning the development of N K cells. Ikaros-1, Ets-1, Id-2, Notch-1 and Jagged-1 have all be implicated in influencing lymphoid cell development. It is these genes that w i l l be further elaborated upon. 1.5 (a) Ikaros For differentiation to occur, there must be a co-ordinated program of activation and silencing of genes. Lymphoid cells are no different in that regulation occurs through 15 extracellular and intracellular signals which affect the expression of genes. Ikaros and its family members are zinc-finger nuclear protein transcription factors that influence the development of all lymphoid lineages (Trinh et al. 2001). The Ikaros gene family consists of four members. They are Ikaros, Aiolos, Helios, and Eos. Expression of the first three occurs predominantly in lymphoid cells, where as expression of Eos occurs solely in neural tissue(Haire et al. 2000). Helios expression is restricted to T cells and Aiolos is expressed in most of the same cell types that express Ikaros. The exception to this being that Ikaros alone is expressed in haematopoietic progenitors (Sabbattini et al. 2001). The murine Ikaros gene was originally reported to contain 7 exons that are alternately spliced to produce at least 10 isoforms. The longer isoforms, Ikaros-1, Ikaros-2 and Ikaros-3 contain three or more N-terminal zinc fingers, and bind D N A . A l l other forms have less than three N-terminal zinc fingers and do not bind D N A . Ikaros splice variants interact with each other either in homodimers, or heterodimers via two zinc fingers in a common C terminal domain. Binding of an isoform with one that is D N A non-binding w i l l result in a dominant negative phenotype, where the pair w i l l be unable to bind D N A (Payne et al. 2001). Ikaros-1 is the largest isoform, with four zinc-finger domains close to the N-terminus. The Ikaros gene is essential for T, B and N K cell development. Knock out (Ikaros -/-) mice lack B cells, N K cells, and fetal T cells (Sabbattini et al. 2001). Ikaros proteins which are mutated to specifically not contain D N A binding domains, result in more severe lymphoid cell defects. Mice die 1-3 weeks after birth. A hypothetical reason for this may be in that these mutants can still heterodimerize with other family members (such as Aiolos and Helios), interfering with their activity (Cortes et al. 1999). 16 There are a number of mechanisms by which Ikaros could affect gene expression. Binding sites for Ikaros have been located in certain lymphoid gene promoters. This implies that Ikaros acts as a classical transcription factor (Sabbattini et al. 2001). Further studies have elaborated on this hypothesis by showing Ikaros' association with transcriptional machinery (Cortes et al. 1999). Ikaros is though to act as both an activator and a repressor of gene expression. Evidence that implicates Ikaros as an activator comes from biochemical studies which have isolated Ikaros proteins with components of chromatin remodelling complexes containing the molecule SWI/SNF. Upon T-cell activation, the SWI/SNF complex is targeted to chromatin and participates in its decondensation which results in gene expression. Ikaros is thought to target the SWI/SNF complex to the repressed chromatin (Cortes etal . 1999). There is compelling evidence that implicates Ikaros as a transcriptional repressor as well . Firstly, Ikaros has been shown to target the M i - 2 - H D A C complex to heterochromatin to preserve its deacetylated state. A s well , Ikaros has been shown to target the m S i n 3 - H D A C complex to more accessible chromatin regions to provide a short-term repressed state (Cortes et al. 1999). The action of these different Ikaros containing complexes may influence the fates of lymphoid cell differentiation. Another line of thought suggests Ikaros contributes to heritable gene inactivation by recruiting genes to foci of pericentromeric heterochromatin (Trinh et al. 2001). Recent studies focus on the idea that Ikaros may help to recruit genes to these pericentromeric heterochromatin regions. One study showed in T lymphocytes, interaction of Ikaros and the TdT promoter was essential for gene down-regulation. Chromatin structure of the 17 TdT promoter was altered, then pericentromeric repositioning was thought to occur (Trinh etal . 2001). The role of Ikaros in lymphoid cells seems to be that of activator/repressor. There also seems to be many mechanisms by which Ikaros exerts its effects. Its involvement in N K cell development is less understood, but knock-out studies prove it to be an essential protein for their development. 1.5 (b) Ets Since it was first cloned, the Ets transcription factor has had 30 members of its family identified. They have been identified in a wide range of species ranging from insects to humans (Maroulakou and Bowe 2000). The Ets transcription factor contains a winged helix-turn-helix D N A binding domain. A l l members of this large family bind to a unique G G A A / T D N A sequence called the Ets binding site (EBS). The location of this sequence in viral and cellular gene promoters has implicated its involvement in expression of genes controlling cellular proliferation, differentiation, development, hematopoiesis, apoptosis, metastasis, angiogenesis, and transformation. Over 200 Ets target genes have been identified, and this number continues to grow (Sementchenko and Watson 2000). Ets-1 expression during morphogenesis occurs in non-lymphoid tissue, and in lymphoid tissue including spleen and thymus. During neonatal development it is interesting to note that Ets-1 expression is high in regions of the thymus. This high level of expression is maintained in a certain subset of adult tissue including spleen and thymus (Maroulakou, Bowe 2000). Ets-1 expression occurs in B , T, and N K cells of adult mice. While its expression is not required for the development of B and T lineages, as shown 18 through knock out studies (Barton et al. 1998), it appears to be essential for their survival and maturation. More pertinent to the focus of this thesis, Ets-1 is required for the development of functional N K cells in mice (Maroulakou, Bowe 2000). Gene targeting studies in mice created a null mutation of the Ets-1 gene resulting in the conclusion that this transcription factor is essential for the development of mature N K cells in mice. Ets-1 deficient mice displayed severely reduced or absent N K cell function in vitro and in vivo. They were unable to display cytolytic activity against either tumour cell and class I M H C deficient targets. This function could not be rescued by culturing Ets-1 ~'~ bone-marrow with exogenous 11-2,11-12, or 11-15. The mechanism of Ets is such that family members often differ in their exact binding site preference outside of G G A A / T core, with factor specific recognition spanning nine to fifteen base pairs. It is the sequence geometry in relation to other cis-elements that controls the target gene specificity and affinity of Ets factors. These neighbouring regions of D N A form complexes and act synergistically with members of other transcription factor families (Sementchenko, Watson 2000). Most Ets proteins can bind D N A as a monomer and binding is often enhanced or modulated by other factors. A synergistic relationship is illustrated by the binding of Ets-1 - C B F - D N A complex. While C B F increases the binding affinity of Ets-1 to D N A , Ets-1 decreases the rate of dissociation of C B F from the T-cell receptor a and p enhancers ( L i et al. 2000). Most of the Ets proteins have been established as transcriptional activators. In the last few years however, an increasing number of family members have also been identified as transcriptional repressors (Mavrothalassitis and Ghysdael 2000). It is the 19 interaction between Ets and other transcription factors that result in either activation or repression ( L i et al. 2000). The protein Ets-1 specifically has been shown to be essential in all lymphoid lineages. It is involved in B cell maturation, T cell activation, and N K cell development and function (Barrel et al. 2000). While the family is large and each member plays a slightly different role, Ets-1 is involved in gene activation, not repression (Mavrothalassitis, Ghysdael 2000). Due to high expression levels in adult N K cells, Ets-1 may participate in N K cell gene regulation required for normal development. In section 1.4, it was noted that in the murine fetus, N K cell development occurs in the thymus. Since Ets-1 levels are highest in the thymus during neonatal development, it is plausible that Ets plays a role in developmental gene regulation in the N K cell lineage. 1.5 (c) Id-2 Basic helix-loop-helix (bHLH) transcription factors, especially ones in the E protein family, are involved in controlling differentiation of lymphoid lineages. There are two conserved domains in b H L H proteins. The H L H domain mediates protein-protein interaction, and the basic domain allows for binding of b H L H proteins to D N A sequence in a target gene (Spits et al. 2000). A family of inhibitors of D N A binding proteins, called Id, regulates transcriptional activity of b H L H proteins. There are four members, Id 1-4, which have highly homologous H L H domains, and have differential tissue distribution (Heemskerk et al. 1997). The H L H domain of Id can interact effectively with b H L H proteins, binding them and preventing them from binding D N A rendering them transcriptionally inactive (Spits et al. 2000). 20 The determining factor in certain cell lineage decisions is dependant on the dose of Id and b H L H proteins present. In B cell and T cell development, the E proteins play a part in determining lymphoid precursor fate. When Id2 and Id3 are overexpressed, T and B cell development is inhibited. N K cell development is however, is not affected, and it is hypothesized that Id proteins allow N K cell development by inhibiting alternative lymphoid precursor pathways (Spits et al. 2000). M i c e lacking Id2 display a marked reduction in N K cell population. E proteins lead to T cell lineage, but when they are bound and inactivated by Id2, bipotent T / N K cell progenitors differentiate into N K cell lineage (Ikawa et al. 2001). These bipotent progenitor cells are present at normal numbers in the Id2 -/- fetal mouse. Therefore Id2 works as a switch on these bipotent progenitor cells after they have developed, affecting their differentiation. Id2 mutants are unable to support bipotent progenitors differentiating to N K cells, consequently resulting in them becoming T cells (Ikawa et al. 2001). It is important to note that fetal N K cell differentiation occurs from T / N K cell precursors, in the thymus (as mentioned in section 1.4 a). A s indicated above, other precursors to N K cells must exist as N K cells are present in athymic mice. Id2 may not only work in the fetal thymus at the level of regulating the T / N K progenitor cell. It may also regulate the progenitor to N K cells found in the bone marrow later in murine development (talked about in section 1.4 a). Evidence for this suggestion comes from the finding that Id2 is expressed in progenitor cells found in bone marrow (Rosmaraki et al. 2001). Another population of cells, C D 4 + CD3" Il-7Roc+, which are key players in the formation of Peyer's patches, can be induced to differentiate into N K cells in vitro. This 21 population of cells is not present in fetal thymus and they do express Id2 (Yokota et al. 1999). The combined studies all illustrate a common thread. Id2 is an important transcription factor in the development of N K cells, and further characterization of it can shed light on mechanisms underlying N K cell differentiation. 1.5 (d) Notch-1 and Jagged-1 There are four vertebrate Notch genes (notch-1 - 4). These genes encode a highly conserved family of large type I transmembrane glycoprotein receptors. There are a series of important domains in these receptors which lends function to the Notch receptor. The extracellular epidermal growth factor-like (EGF) repeats are involved in ligand binding. The intracellular ankyrin-like repeats are required for downstream signalling (Pui et al. 1999). There are multiple ligands to Notch proteins. In Drosophila they are Delta and Serrate, and in mammals Delta homologs are called Delta-like and Serrate homologs are called Jagged (Osborne and Miele 1999). The Jagged family consists of Jagged-1 and 2. The Delta-like family is comprised of Delta-like-1, and 3 (Deftos and Bevan 2000). A s well as containing EGF- l ike repeats in extracellular domains, Notch ligands have an important cysteine-rich N-terminal domain (DSL domain) which is responsible for Notch binding. In mice, targeted disruption of Notch-1, Notch-2, Jagged-1 or Jagged-2 results in embryonic lethality or severe developmental defects (Osborne, Miele 1999). The mechanism underlying Notch interaction with its ligand begins by a proteolytic cleavage of Notch upon binding. This cleavage occurs within or close to the transmembrane domain of Notch and results in the intracellular domain releasing and 22 translocating to the nucleus of the cell. The intracellular domain contains a R A M sequence that interacts with C B F 1 transcription factor, and it contains six ankyrin repeats that are essential for activating Notch dependent gene expression. The interaction with C B F 1 converts it from a repressor to an activator of gene transcription (Deftos, Bevan 2000). Notch-1 has been implicated as a main character involved in lymphoid cell fate determination. It is found in C D 3 4 + hematopoietic precursors, and its ligand Jagged-1 is expressed on bone-marrow stromal cells and thymic stromal cells. There is evidence to suggest that Notch-1 influences the choice between C D 4 and C D 8 T cell lineages. A s well , mice heterozygous for Notch-1 expression resulted in an accumulation of y8 T C R lineages as opposed to the normally seen larger number of a(3 T cell lineages. Notch-1 is also thought to play a role in the lineage choice between B and T cells. Evidence exists to suggest that early lymphoid precursors, in the absence of Notch-1 (via inducible inactivation) choose a B cell fate in bone-marrow independent fashion. Constitutively active Notch-1 results in T cell lymphomas and no B cell development (Osborne, Mie le 1999). Notch interaction with its ligand can result in a CBF-1 independent signalling pathway as well . Intracellular Notch may bind a conserved zinc finger cytoplasmic protein called Deltex. The result is repression of b H L H protein E47. E47 when bound as a homodimer favours B cell commitment, however E47 bound as a heterodimer influences T cell development. It is thought that Notch-1 represses the homodimer formation of E proteins and thereby represses B cell development (Osborne, Miele 1999). Studies have shown Notch-1 represses E47 resulting in a block in B cell development and a drive to T cell 23 development from precursors. In section 1.5 (c) it was described how Id-2 is known to bind and inhibit the E proteins thereby promoting an N K cell fate. While the link between Notch, Jagged and N K cell development is not obvious, there are clues in the evidence presented that imply Notch may inadvertently play a role. Through deltex signalling pathways lymphoid progenitors may be pushed to the T / N K cell lineage (as Notch-1 represses B cell formation from these progenitors). These T / N K progenitors may be then influenced via other factors (such as Id-2), which may direct these cells to the N K cell lineage. 1.6 Thesis Objectives Our laboratory developed an in vitro system whereby ES cells were cultured with specific cytokines to differentiate into N K cells. These cells are phenotypically similar to N K cells, however the N K - l i k e cell is not able to express the Ly49 family of inhibitory receptors. The genes involved in regulating the acquisition of the Ly49 family of receptors are unknown, as are the genes are involved in the proper development and maturation of the N K cell. It is very difficult to isolate large numbers of N K cells at different stages in development in vivo for the purpose of this study. It was the goal of this project to characterize the expression patterns of five chosen genes throughout the in vitro development of the N K cell. Specifically, the objectives were to determine 1) whether expression of each gene was on or off at five different time points along the developmental path of the N K cell, 2) whether there was some semi-24 quantitative difference between the expression on different days observed for a particular gene and 3) whether a difference could be seen between the N K - l i k e cells and L A K cells. 2 5 Chapter 2 Materials and Methods 2.1 Oligonucleotides 2.1(a) Oligonucleotide design P C R primers as well as oligonucleotide probes were designed for each gene using a mouse c D N A sequence obtained from N C B I (National Centre for Biotechnology Information). They were synthesized by Life Technologies Inc. The sequence of all the P C R primers and the oligonucleotide probes are listed in table 2.1. 2.1(b) Oligonucleotide specificity Once P C R primers and oligonucleotide probes were designed, the sequence was put into a B L A S T seach to confirm specificity of each oligonucleotide to the gene studied (as each gene is either part of a gene family, or has splice variants). 2.2 Control Tissues 2.2 (a) Spleen Single cell suspension was prepared from the spleen of an eight week old B6 mouse using a 70 (im nylon cell strainer (Falcon). Red blood cells were then lysed by adding 4 ml of 0.83% N H 3 C 1 , vigourous pippetting, and incubation for 5 minutes at room temperature. The mixture was neutralized by adding R P M I media, and the cells were then Fig 2.1 PCR Primers Primer Name PCR product size Primer Sequence mJagged1#2-5 ' 900 bases C T G T G T G G A T G A G A T CAA T G G C T A mJagged1#2-3 ' G A C C G T G T T G G C T C C G T G T T T C mld2-5 ' 733 bases T G G A C G A C C C G A T G A G T C T G C T mld2-3 ' A C C G A A G A C T T C A T T T A A G T A A C C G m Notch 1-5' 622 bases G C A G T C T C C A T C C A T G C C T C T C m Notch 1-3' C C A G C G G T T G T A C A T C T G C C T G mlkaros-5 ' 850 bases G A C C T G T G C A A G A T A G G A G C A G mlkaros-3 ' G G C A T A A C C A G C T A T C T T T G T G C mEts-5 ' 991 bases G G T G A T G T G G G C T G T G A A T G A G T mEts-3 1 C G T A G C G C T T G C C C G C C G T C m G A P D H - 5 ' 494 bases G T T C C A G T A T G A C T C C A C T C A C G m G A P D H - 3 ' G T G G A T G C A G G G A T G A T G T T C T G 2.2 Oligonuleotide Probes Gene Oligo Name Oligo Sequence Jagged-1 f rom m o u s e mJagged-Ol igo T C T A C A T A G C C T G T G A G C C T T C C Id-2 f rom m o u s e mld-2-Ol igo C C T G C A G C A C G T C A T C G A T T A CA Notch-1 f rom m o u s e m N o t c h l -Ol igo C A G C A G C C T C T C C A C C A A T A C C Ikaros-1 f rom m o u s e mlkaros-Ol igo A G C T C C A T G T A C C A G C T G C A C A A Ets-1 f rom m o u s e mEts-Ol igo T G T C C C T C A A G T A T G A G A A C G A C T G A P D H f rom m o u s e m G A P D H - O l i g o T C C C T G C A C C A C C A A C T G C T T A G C 27 centrifuged (Heraeus Instruments Inc) for 5 minutes at 1500 R P M . The supernatant was removed and the cells were washed twice with 15 ml of R P M I . Cells were counted and adjusted to be at a concentration of 5-10 x 10 7 cells/ml. 2.2 (b) Thymus Thymus was isolated from an eight week old B6 mouse. The tissue was cut up into small pieces using sterile scissors. The tissue was further sheared using a 21 gauge needle in a 3cc syringe, by sucking up and expelling the mixture several times. The mixture was then put into a 15 ml falcon tube and left for several minutes to let the larger particles settle. The cell suspension was then transferred to a new 15 ml falcon tube. Cells were counted and adjusted to be at a concentration of 3-6 x 10 7 cells/ml. 2.2 (c) Bone Marrow The femur and tibia of an eight week old B6 mouse were removed and cut at their ends. A 26 gauge needle was used to suck up the media from the culture dish and to inject the media through the hole in the bone to flush the bone marrow into the culture dish. Contents of the culture dish were added to a 15 ml falcon tube and larger particles settled at the bottom. Then the cell suspension was transferred to a new falcon tube. Cells were counted and adjusted to be at a concentration of 4-6 x 10 7 cells/ml. 28 2.3 Ce l l Culture 2.3 (a) Culture System There are three main stages in the protocol that differentiates ES cells into N K cells. Firstly ES cells were trypsinized , then resuspended in Iscove's Modified Dulbecco's Medium ( I M D M ) . This was added to a methylcellulose media which contained 15% F B S , 2 m M L-Glutamine, 150 u M M T G , 40ng/ml stem cell factor (SCF) and 20 ng/ml vascular endothelial growth factor, and dispensed at a concentration of 350 cells/ml into 35 mm Petri dishes (Stem Cel l Technologies, Vancouver, B C ) . These plated cells were incubated at 37°C and 5% CO2 for a period of 8 days. The clusters of cells that formed were washed to remove methylcellulose agar and trypsinized. B y passing cells through a 21-gauge 1 '/2-inch needle three times, they were made into a single cell suspension. Lastly to isolate the C D 3 4 + cells, the suspension was stained with anti-CD34-FITC m A b and sorted on the FACStarplus ™. In the second stage, C D 3 4 + cells were seeded on an OP9 stroma in 6-well plates at a concentration of 10 4 cells per well and cultured for 7 days with 30ng/ml IL-6, IL-7, 40ng/ml S C F and lOOng/ml Flt3-ligand. Three days later, half of the media was removed and fresh medium containing the same growth factors was added. Two days later, the cells were trypsinized, vigorously pipetted, washed and transferred to a new culture of OP9 stroma. In the third stage, the growth factor medium was replaced with fresh medium containing 1000 U / m l IL-2, 20 ng/ml IL-15, 20 ng/ml IL-18 and 1 ng/ml IL-12. The developing ES cells were incubated in this cytokine cocktail for 7 days, with a transfer to a new OP9 29 stromal layer after the first 3 days. On day 8, differentiated ES cells were harvested by vigorous pipetting for analysis. 2.3 (b) Staining and Sorting A t 5 different stages along the developmental pathway of an N K cell in this culture system (fig 2.6), cells were obtained, and centrifuged at 3000 R P M for 3 minutes. Supernatant was discarded and 50 p:l of 2.4G2 antibody was added to block the Fc receptor. This was left on ice for 15 minutes. To the 50 u.1 suspension, 1 | i l of F I T C conjugated anti-Ff-2K b antibody was added. The cell suspension was incubated on ice in the dark for 30 minutes, then 200 u.1 P B S 2% F B S was added to the tube. The contents were transferred to a small falcon tube. Another 200 ui of P B S 2% F B S was used to rinse out the original tube to remove any remaining cells. This 400 (il cell suspension was put on ice, and covered with tin foil. The cells were sorted into a 96 well plate (Falcon) that had 50 u.1 of GIT (guanidine isothiocyanate) which lysed them. Cells were sorted into 10 cells x 3 wells, 100 cells x 3 wells, and 1000 cells x 3 wells by the FACStarplus™ 2.3 (c) Culture of L A K cells Spleen cells were isolated from a 6 week old mouse. The following day, the non-adherent cells were harvested and re-plated in new cell culture dishes for 3 days with IL-2 lOOOU/ml. Adherent cells were then collected and suspended in 1ml wash ( R P M I + 5%FBS) . The cell suspension was transferred to appropriate F A C S analysis tube (Falcon), and then centrifuged at 1500 R P M for 10 minutes. The supernatant was poured 30 off and 50 u,l of 2.4G2 antibody was added. This was left on ice for 15 minutes. 1 u.1 of F ITC conjugated anti-CD3 , and 1 | i l of P E conjugated a n t i - N K l . l were added to the tube. This was left in the dark on ice for Vi hour. After this incubation, 1 ml of PI (propidium iodide) buffer was added to sample. The cells were then sorted for N K 1 . 1 positive C D 3 negative cells into 100 and 1000 cell alliquots. 2.4 R T - P C R 2.4 (a) R T - P C R for controls Total cellular R N A from the above cell suspensions of spleen, thymus, and bone marrow was prepared by adding 1 ml of Trizol Reagent (Life Technologies) to each suspension. R N A was purified according to the manufacturer's protocols. R N A was dissolved in 15 u l of D E P C - H 2 0 (0.1% Diethyl pyrocarbonate) (Aldrich Chemical Co.). Synthesis of c D N A was done using eady-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech) According to the manufacturer's protocols. The primers used were 0.5 pig oligo (dT) (Life Technologies) P C R amplification of c D N A was performed in a 50 u i reaction volume containing c D N A , l ( l M of primer specific to each gene (Fig 2.1), 1.5 p i of 50mM M g C l 2 , 1 u l of l O m M dNTP mix, 5 units of D N A Taq polymerase (Life Technologies), and 1 u.1 of lOx P C R buffer (Life Technologies). First, thirty-five cycles in a GeneAmp 9700 fhermocycler (PE Applied Biosystems) were carried out as follows: denaturation of 30s at 94°C; annealing of 30s at 58°C; extension of 2 min at 72°C; followed by a 7 min hold at 72°C. To optimize conditions for the genes, a second P C R was done with less cycles and 31 higher annealing temperature. Thirty cycles in a GeneAmp 9700 thermocycler (PE Applied Biosystems) were carried out as follows: denaturation of 30s at 94°C, annealing of 30s at 62°C, extension of 2 min at 72°C, followed by a 7 min hold at 72°C. For ETS-1 , the annealing temperature of 60 °C worked best. For all P C R products, D N A was loaded onto a 1% agarose gel (Life Technologies) stained with ethidium bromide, and was run at 80 volts for 2 hours (Fig 2.3). 2.4 (b) R T - P C R For a Limit ing Number of Hematopoietic Cells Cells were lysed in GIT, and R N A was precipitated by adding 25 u.1 of 7.5 M ammonium acetate containing 40 u.g of glycogen and 2 volumes of 95% E t O H (ethanol) made with D E P C water. This was stored at -20°C overnight. The mixture was centrifuged (Heraeus Instruments Inc) at 13 000 R P M for 10 minutes at 4 °C. The pellet was washed twice with 100 ill of 70% D E P C - E t O H , then air-dried. After the ethanol evaporated, the pellet was re-suspended in 6 u.1 of D E P C - H 2 0 . To this, 3.5 u.1 of c D N A mix was added [2u.l 5x R T buffer ( B R L ) , 1 u.1 0 .1M D T T ( B R L ) , 0.2 u.1 25 m M dNTPs (500uM), 0.2(il oligo dT at 1 u.g/u.1, 0.1 | i l R N A s e (10U/U.1) (BRL)] . The mixture had 0.5 ul Superscript II (200U/ml) added and was then incubated for 1 hour at 42°C. c D N A was precipitated by adding 5 u.1 of 7.5 M ammonium acetate, 30 u l of 95% D E P C - E t O H , and 2 ul of polyacrylamide carrier (40mM Tr i s -HCl , 20mM N a Acetate p H 7.8, I m M E D T A pH7.8, 1/100 vol of 10% ammonium persulfate, 1/1000 vol T E M E D , leave 30 min, add 2.5 vol E t O H , centrifuge, re-disolve pellet in 20 vol water by shaking overnight). Pellet was washed once with 70% E t O H , air dried, and re-suspended in 5 u.1 of Tailing solution (lu.1 5x Tailing buffer, 0.5ul 100 m M d A T P , 3.5LI1 distilled water). When pellet was 32 Fig 2.3 PCR control conditions Gene PCR annealing temp cycles hybridization temperature j agged-1 55 degrees 35 58 degrees Id-2 62 degrees 30 58 degrees Notch-1 62 degrees 30 58 degrees Ikaros-1 62 degrees 30 58 degrees ETS-1 60 degrees 30 58 degrees Act in 58 degrees 25 58 degrees Fig 2.4 PCR sample conditions Gene PCR annealing temp cycles positive controls negative controls j agged-1 55 degrees 35 bone mar row H 2 0 Id-2 62 degrees 30 thymus H 2 0 Notch-1 62 degrees 30 thymus H 2 0 Ikaros-1 62 degrees 30 spleen H 2 0 ETS-1 60 degrees 30 spleen H 2 0 G A P D H 60 degrees 25 sp leen/ thymus H 2 0 fig 2.5 Oligonucleotide Melting Temperature Equation 81.5 + 16.6 (Log [Molar Na+]) + 41 (%GC) - 675/ Primer length Molar Na+: 6 x S S C = 1M % G C : interger value f CfQ as 55' 2 > - CD § cro o o o o CD • tit 2 o CD 3 3 CD •a 0 rt P I 05 03 i—» • o 3 + < m + •< -n 2. o IA tn CO O r- * H I H i CJ1 H i 00 < CO C L Day 0 / CD34+ - Day 6 common lymphoid progenitor - Day 8 Day 11 Day 14 / NK cell 34 dissolved, 0.5 | i l of TdT enzyme ( B R L ) was added and the mixture was incubated at 37°C for 15 minutes, followed by 10 minutes at 70°C. To the 5 (ll of c D N A , 38.75 u l of P C R mix was added [25ul of 2x buffer (20mM Tris p H 8.8, lOOmM KC1, l O m M M g C l 2 ) , 4 u l oligo dT ( lug /u l in Tris p H 7.5), 0.5 u l nuclease free B S A (lOug/ul), 0.25 ul triton xlOO, 5 u l water, 2 u l dNTP mix (except dATP) at 25 m M each (final concentration to I m M of each), 1 u l Taq polymerase (5U/ml)]. Forty cycles in a GeneAmp 9700 thermocycler (PE Applied Biosystems) were carried out as follows: 1 cycle for 1 minute at 94°C, 2 minutes at 37°C, and 10 minutes at 72°C. Then, denaturation for 1 minute 94°C, annealing for 2 minutes 55°C, extension for 10 minutes at 72°C. 2.4 (c) R T - P C R for Five Genes of Interest P C R was performed on 4 alliquots of c D N A obtained from 10, 100 and 1000 cells. It was performed for each of the five genes plus G A P D H as a control, and it was done specifically on the c D N A obtained from the selected time points of the N K cell system (fig 2.6). Analysis of gene expression was also done on 100 L A K cells and 1000 L A K cells. P C R was performed in a 50 ul reaction volume containing: 2ul c D N A ; l u M of primer specific to each gene (fig 2.1); 1.5 u l of 50mM M g C l 2 ; 1 u l of l O m M dNTP mix; 5 units of D N A Taq polymerase (Life Technologies); and 1 u l of lOx P C R buffer (Life Technologies). Some reaction conditions varied according to each gene (fig 2.4). OP-9 cells were analysed for Id-2, Jagged-1, Notch-1, Ikaros-1 and Ets-1 expression to confirm that amplification of these genes in the developing system was not a result of OP-9 contamination. OP-9 total R N A was converted to a c D N A sample and this was 35 diluted 1000 and 10 000 times. P C R on 2ul of each diluted sample was performed at denaturation of 30 seconds at 94°C; annealing of 30 seconds at 58°C; extension of 2 min at 72°C; followed by a 7 min hold at 72°C. This was run for 25 cycles. G A P D H primers were used as a positive control. 2.5 P Labelling of Oligonucleotides For each oligonucleotide in F ig 2.2, 10 ng was labelled using 3 2 P . After heating the oligonucleotide to 65°C, 5 u l of 3 2 P dCTP, 1 u.1 of lOx B S A (NEB) , 1 u.1 TdT enzyme (Gibco), 4 u l TdT buffer (Gibco), and 9 u.1 of d d H 2 0 were added to it. This was incubated at 37°C for two hours. 80 u.1 of dd tkO was added. The sample was purified using a MicroSpin G-25 Column (Amersham Pharmacia Biotech) for 1 minute at 2800 rpm. 2.6 Hybridization 2.6 (a) Controls P C R products for each control tissue (bone marrow, spleen, and thymus), derived from using primers for Jagged-1, Notch-1, Id-2, Ets-1, and Ikaros were run on a 1% agarose gel (Life Technologies) and stained using efhidium bromide. The gel was run at 80 volts for 2 hours. Products were transferred onto a Zeta-probe G T nylon membrane (Bio Rad) using the alkaline blotting method. Hybridization of each 3 2 P labelled oligonucleotide (fig 2.2) was performed overnight at 58°C in 6 x SSC, 0.5% SDS and 0.5% skim milk. The membrane was washed at 20°C below the melting temperature of each 36 oligonucleotide used (fig 3.5), 3 x 1 0 minutes with 3 x SSC and 0.1% SDS and autoradiography was performed for 48 hours, 24 hours, 8 hours, and 1 hour and 15 minutes. Optimal exposure time for Ikaros-1 and Notch-1 was overnight. For ETS-1 it was 1 hour and for Id-2 it was 15 minutes. 2.6.(b) Hybridization of Oligonucleotides Specific For Genes of Interest P C R products derived from using primers for Jagged-1, Notch-1, Id-2, Ets-1, Ikaros-1 and G A P D H were run on a 1.5% 450ml agarose gel at 200 volts (Life Technologies) and stained using ethidium bromide (amounts of c D N A run in fig 2.4). Products were transferred onto a Zeta-probe G T nylon membrane (Bio Rad) using the alkaline blotting method. Each 3 2 P labelled oligonucleotide (fig 2.2) was added (see section 2.2(f) on labelling). Hybridization was performed overnight at 58°C in 6 x SSC, 0.5% SDS and 0.5% skim milk. The membrane was washed 3 x 1 0 minutes with 3 x SSC and 0.1% SDS at 20°C below oligonucleotide melting temperature (fig2.5). The radioactive membrane was exposed for several differing times to film and autoradiography was performed. 2.7 Exposure to Phosphoimager Each radio-labelled membrane containing c D N A from the five genes of interest plus G A P D H was exposed to the phosphoimager and subsequent volume quantification was analized using ImageQuant software. The software calculates the volume under the surface created by a 3-D plot of the pixel locations and pixel intensities. A local average 37 background correction method was used. Here the program determines the average (mean) of all the pixel values in the object outline and uses this value for background. To calculate volume, ImageQuant subtracts the background value from the intensity of each pixel in the object, and then adds all the values. For each gene of interest the volume was normalized by dividing the volume attained for each sample by the volume attained for that same sample when amplified with G A P D H . This way sample volumes could be compared to one another and differences seen in time points would not be due to differences in c D N A amount. 38 Chapter 3 Results The analysis of gene expression was done by P C R and phospho-imaging. P C R specific for G A P D H , Id-2, Jagged-1, Notch-1, Ikaros-1, and Ets-1 was performed on 4 aliquots of c D N A from 10 cells, 4 aliquots of c D N A from 100 cells, and 4 aliquots of c D N A from 1000 cells. The c D N A was obtained from different days along the in vitro development of an N K cell (days 0, 6, 8, 10, and 14) (Fig 2.6). ES cells were cultured into embryoid bodies which are clusters of cells (a small fraction being CD34+). Embryoid bodies were harvested and stained with anti-mouse CD34 m A B . The CD34+ embryoid body (day 0 cells) were seeded onto a confluent OP9 layer and over a 14 day period, they were exposed to different cocktails of cytokines which influence their developmental pathway (Fig 2.6). The culture system was maintained by Moto i Maeda and Rebecca Lian. A t each time point, R N A was isolated from aliquots of 10, 100 and 1000 cells and reversed transcribed into c D N A . This c D N A was P C R amplified and then used to investigate gene expression patterns. Analysis of gene expression was also done on 100 L A K cells and 1000 L A K cells. The resultant amplified D N A was run on a gel and transferred to a membrane where it was hybridized by P 3 2 labelled oligonucleotide probes. Membranes were then analyzed by phospho-imaging. Audioradiography confirmed that the bands that hybridized to labelled probes were the same bands shown in the gel pictures (data not shown). OP-9 cells were analysed for Id-2, Jagged-1, Notch-1, Ikaros-1 and Ets-1 expression to confirm 39 that amplification of these genes in the developing system was not a result of OP-9 contamination. 3.1 Oligonucleotide Design Id-2, Notch-1, Jagged-1, Ets-1, and Ikaros-1 are five genes known to play a role in N K cell development. In order to determine the expression of these genes, primers and oligonucleotides were designed, and P C R conditions were established. To detect the expression of each gene of interest by R T - P C R , c D N A sequences were obtained for each gene from N C B I and primers and oligonuleotide probes were designed from this sequence (Appendix A ) . Oligonucleotide sequences were put into a B L A S T search to confirm that they were specific for Id-2, Jagged-1, Notch-1, Ikaros-1 and Ets-1. Bone marrow, spleen and thymus were investigated as possible positive controls for each set of primers designed for the 5 genes of interest. After investigating this via literature, these three tissues were used to test expression of the genes. c D N A from each of these tissues could potentially serve as a positive control for future experiments. F ig 3.1 illustrates the P C R products (5 genes of interest) amplified in bone marrow, spleen and/or thymus. Product indicates that the bone marrow, spleen or thymus c D N A could then be further used as a positive control in subsequent P C R analyses. Id-2, Ikaros-1, and Ets-1 were amplified in each of bone marrow, spleen and thymus indicating that c D N A from any o f the three could be used later as a positive control for the gene of interest. Thymus was chosen as the control c D N A for Id-2, and spleen was chosen for Ikaros-1 and Ets-1. Jagged-1 was only amplified in bone marrow and consequently, this was the 40 Id-2 (733) Jagged-1(900) Notch-1(622) 600 B S T 600 600 B S T B S T Ikaros-1(850) 600 EtS-1(991) 600 B S T B S T F ig 3.1 Gel pictures of bone marrow (B), spleen (S) and thymus (T) which were each tested for gene expression using primers for each of the 5 genes of interest. A 100 base pair size marker was used. The position of 600 bases is indicated on each figure. The size of the amplified gene is indicated next to the gene name. Each P C R condition was unique to each gene. 41 c D N A used as a positive control for Jagged-1. Notch-1 was amplified in spleen and thymus, the latter being chosen as the positive control for Notch-1. 3.2 G A P D H The amplified c D N A from each time point was analyzed for G A P D H expression. In all the samples, G A P D H c D N A was detected indicating successful R N A extraction, c D N A synthesis, and the first round of P C R to amplify total c D N A . Generally, the amount of P C R products increased as more cells were used. Although day 6 and day 10 may show some discrepancy, the overall pattern indicates that the concentration of c D N A increases with number of cells respectively (Fig 3.2). When quantified, some samples had a concentration of c D N A in 100 cells that appeared to be higher than the concentration of c D N A in 1000 cells (data not shown). This is irrelevant, as these values w i l l be used to normalize the values given for the 5 genes of interest. 3.3 Id-2 The pattern of expression of Id-2 did not change markedly during in vitro N K cell development. Its expression was detected at every time-point during the 14 day culture (Fig.3.3 a). The gel picture (Fig 3.3 b) is quite similar to the phospho-image shown in F ig 3.3 (a). Upon analysis of the phospho-image southern blot, it is apparent that expression of Id-2 is detected in almost every aliquot tested and at every time point investigated (Fig 3.3 c). Ten cell aliquots tested for day 8 and day 14 revealed expression of Id-2 in only 3 of the 4 samples tested. Overall it is fair to say that the frequency of Fig 3.2 G A P D H 42 Day 0 Day 6 10 100 1000 10 100 1000 494 bases Day 8 Day 10 10 1000 10_ \ f 100 1000 Day 14 L A K o o o o c£ £ T2. T2. o o o o S B 22 o o g a g g Fig 3.2 Phospho-image of c D N A derived from either 10, 100, or 1000 cells that were tested for G A P D H expression at different time points. The + indicates positive controls used, and the - indicates a negative control of H 2 0 . The size of the P C R product is indicated on the left side of the image. Fig 3.3(a) Id-2 43 DayO 10 100 1000 10 Day 6 100 1000 733 bases Day 8 10 ~\ r 100 1000 \ I r~ 10 Day 10 100 ^ 1000 L A K Fig 3.3(b) 10 5* ** T Fig 3.3 (a) is a phospho-image of PCR amplified ID-2 cDNA. The size of the fragment is indicated on the left of the figure. Figure 3.3 (b) is a gel picture of the PCR amplified cDNA. The dark band adjacent to the * is the position of the 600 base band of a 100 base pair ladder that was run on either end of the gel. The first four wells of each day is PCR amplified c D N A from 10 cells. The next four wells is PCR amplified c D N A from 100 cells, and the last set of four wells from each day is PCR amplified c D N A from 1000 cells. The + indicates a positive control of either bone marrow (B), spleen (S) or thymus (T). The is a negative control of H 2 0 . L A K indicated PCR amplified c D N A from 100 and 1000 L A K cells respectively. 0001 ^ ou^  >. 111 O H H t a i 0L Q i t i i f o 0001 ->\ ' 1 * 1 1 0 0 1 £ 1 1 j 01 000 L % CD I ! 1 001. Q 01. - 0001. % CD 001. Q t 1 1 01-- oooi. ° I | i CD 001. Q • 1 1—1—1 01-•^l- CO CM T - O spueg }0 jaqiunN o Pi, lit i=L s l l s o 0001 is i i^ooi >• CO Q c c s||« 001 0001 s||«00L >, Q s||»0l sil^o 0001 oo s||«001 >. cd Q S||eo oi s | | « 0001 sneooOl «' s||so oi siiso 0001 SUM 001 j s Q S | | 3 O 0 l CN i n m o d (|OJ)uo3/3|diues) A^sua)U| pueg co on *j CO s 'o 2 ft S Cu) ^ S U • | 3> •S . 1 1 , 3 ~ 0) o cs a, .2 8 8 ft o O T3 U g o <u ,=< T3 » co O c3 C + H aj O w EL =TS o ^2 5 a o O CCS C N i T3 H H <4—i O e _o C O Cu) l-c p< X Cu) O >> o a a> G -6 £ o e ft Cu) C CD Cu) c n "5 S*8 £ 0 0 ts 3 .2? ,3 > 45 expression of Id-2 is quite high. In an attempt to semi-quantify expression levels of Id-2, F ig 3.3 (d) illustrates clearly that day 0 expression is lowest of all the time points tested. Day 6 expression levels of Id-2 seems to rise and on day 8 the levels seem to drop, though not as low as what is shown for day 0. Day 10 shows a slight increase in expression from day 8. Day 14 shows a strange pattern as expression is high for one 10 cell sample and two 100 cell samples. The slight stepladder appearance for 10, 100 and 1000 cells of day 6 and 10, and the strange pattern seen in day 14 is suggestive that while frequency of expression of Id-2 may be high (as suggested in F ig 3.3 c), it is unlikely 100% of the cells expressed Id-2. Ideally i f every cell expressed Id-2 at the same level, the level should be the same for 10, 100 and 1000 cells tested for a specific day as the values are normalized to G A P D H expression. L A K cells were scrutinized carefully in relation to what pattern was shown for day 14 (Fig 3.3 d). Expression of Id-2 in L A K cells appears low and is somewhat consistent with expression shown at day 14. The expression is nearly identical in 1000 cells tested for both L A K and day 14. The difference seen between day 14 and L A K for 100 cells is probably due to using a small sample number in the analysis such as 100 cells. Expression does not occur in 100% of the cells and the 100 cell sample for day 14 shows a skewed result. 3.4 Jagged-1 and Notch-1 The expression pattern of Jagged-1 becomes very interesting when viewed in conjunction with Notch-1 expression pattern. Virtually nothing can been seen from the gel picture of Jagged-1 (Fig 3.4b), yet the phospho-image indicates expression of Jagged-Fig 3.4(a) Jagged-1 46 900 bases D a y 0 1000 D a y 6 -LQ- 1000 D a y 8 -10- 100 1000 D a y 10 _L0- 1 ooo 10 D a y 14 100 L A K 1000 *+ i— o-o o o O O 3 o <o Fig 3.4(b) 10 14 LAIC BM Fig 3.4 (a) is a phospho-image of PCR amplified Jagged-1 cDNA. The size of the fragment is indicated on the left of the figure. Figure 3.4 (b) is a gel picture of the PCR amplified cDNA. The dark band adjacent to the * is the position of the 600 base band of a 100 base pair ladder that was run on either end of the gel. The first four wells of each day is PCR amplified c D N A from 10 cells. The next four wells is PCR amplified c D N A from 100 cells, and the last set of four wells from each day is PCR amplified c D N A from 1000 cells. The + indicates a positive control of either bone marrow (B), spleen (S) or thymus (T). The is a negative control of H 2 0 . L A K indicated PCR amplified c D N A from 100 and 1000 L A K cells respectively. 0001-. O I L 5" 01-0001- ? 001. 2 01-oooi. 001-01-01 oooi. 001-01-^ n CM T- o spueq }o jaqwnN CD Q 0001- % 001 Q CD Q 1 1 T" OiJOl •* 0001 001 01 0001 0i)L 01 0001 00 L 01 oooi 001 01 0001 001 01 m O J m < N r i r in o -; Q ( |OJJUO0/3|dUJBS) A ) jSU3)U| pueg cz> c » . § i .(-> CC3 S SP 41 ™ c/> o en O 3 <u I—I i - l o "5 O c« ,—, o e o c . l l s « o o a S .5 N VH I S o - 1 3 « T 3 'fe a 3 * o 9 " O •35 ^ g 71 u S s *° a r x cu • -g ^ « h c i 5 w CU - 0 ^ 2 | .&p > '£ 3 T3 48 1 occurs on day 0 and day 14 (Fig 3.4a). There are no bands present for days 6, 8, and 10. There is no band present for 100 L A K cells and there is a band present for 1000 L A K cells. The results indicate that Jagged-1 is expressed at day 0, not expressed at day 6,8,10, and expressed again at day 14. Upon observation of Notch-1 expression, it becomes apparent that Notch-1 does not follow the same pattern as Jagged-1, which is intriguing as Jagged-1 is a ligand for Notch-1 in N K cells. Generally, Notch-1 seems to be constitutively expressed through out the development of the N K cell, as bands are apparent in the phospho-image in each time-point observed (Fig 3.5a). Day 6 and day 10 appear brightest as they do in the gel picture (Fig 3.5b). Notch-1 expression was also detected in L A K cells, just as Jagged-1 was. Upon closer examination of the phospho-image it is apparent that for Jagged-1, from 1000 cells analysed, only two bands are present out of 4 samples tested in day 0 and day 14. This pattern shown (Fig 3.4 c) illustrates that Jagged-1 has a very low frequency of expression. Phospho-imager analyses are intended to provide more quantitative information. However, not much definitive information can be gained from F ig 3.4 (d). What can be said is that expression in day 0 is higher than days 6, 8 and 10, and seems to be higher than what is shown for day 14. Although it may appear higher than in day 14, it could actually be a result of a higher frequency of expression in day 0 (as expression is seen in 100 cells tested as well as 1000 cells for day 0) (Fig 3.4 c and d). What is interesting to note is the differential pattern of expression for Jagged-1 compared to Notch-1. Jagged-1 is expressed day 0 and day 14 at a low frequency. Notch-1 is expressed every day tested at a moderate frequency (Fig 3.5 c). Every day tested in all four samples of 1000 cells showed expression of Notch-1. When cell aliquots of less than Fig 3.5(a) Notch-1 49 r 622 bases Day 0 r Day 6 _ A 1 _ F 10 , , 100—^ , 1000 , , 10—^ , 100—^ , 1000-10 Day 8 r - » , — . i n o n i * — — ^ t— 00-Day 10 100 1000 s-Fig 3.5(b) . 10 Fig 3.5 (a) is a phospho-image of PCR amplified Notch-1 cDNA. The size of the fragment is indicated on the left of the figure. Figure 3.5 (b) is a gel picture of the PCR amplified cDNA. The dark band adjacent to the * is the position of the 600 base band of a 100 base pair ladder that was run on either end of the gel. The first four wells of each day is PCR amplified c D N A from 10 cells. The next four wells is PCR amplified c D N A from 100 cells, and the last set of four wells from each day is PCR amplified c D N A from 1000 cells. The + indicates a positive control of either bone marrow (B), spleen (S) or thymus (T). The - is a negative control of H 2 0 . L A K indicated PCR amplified c D N A from 100 and 1000 L A K cells respectively. mm • 11111 IIP s p • oooi. 001-01-to Q 0001- -001- CD Q 01. 0001-001. 00 >* CD Q 01. 0001-001. CO >> CD Q 01. 0001-001. o >> CD Q 01. ^ " W M T- O spueg pi jaqiunN i n OOOI OOOI 001 Q 01 0001 001 01 0001 001 01 0001 001 01 0001 q ooi - i — i - r — r -n in CM in T - in o OJ T — O ( | O j ) u o 3 / 3 | d u i e s ) A)!sua)U| p u e g 01 o co 2 U a SP oo O <U & O co § ^  2 S •« f cd O © "S o g o u e * H co O CO S co ^ E &'» 1 eh 5 0 3 ^ ) CO •3 co G N ^ ^ «a _ i '5 I f £ o o £ 6 ft o j 3 a ^ *3 S C 3 CO .2 o -o co C/3 J3 co ca >» £ § 5*^ « M cS <U w co ^3 U-i O ^ 1? m to ^ 3 E 3 ^ 51 1000 cells were analysed expression was not detected in every sample tested. This indicates that while frequency of expression seems higher than Jagged-1, it is still not occurring in 100% of the cells. The expression level of Notch-1 is low in day 0, yet higher than what is shown for day 6. The one spike in expression shown for day 6 1000 cells is probably due to Notch-1 having a low frequency of expression during this time period. Expression level for day 8 and 14 is quite low, and is higher in day 10. The appearance of a low expression level in day 8 and 14 could be just a result of the low frequency of expression of Notch-1 on these days (Fig 3.5 c) as opposed to actually being a low level of expression. Another interesting observation regarding the differences in expression patterns between Notch-1 and its ligand Jagged-1 is expression in L A K cells. Surprisingly there is very low expression of Jagged-1 in L A K cells, and high expression of Notch-1 in L A K cells. A s well it is worth noting that expression levels shown of Notch-1 in L A K cells are higher than what is illustrated in day 14 cells (Fig 3.5 d). 3.5 Ikaros-1 The transcription factor Ikaros-1 shows a very interesting pattern of expression in the developing N K cell. Firstly, the most obvious observation is that it seems to be expressed throughout the culture period (Fig 3.6a). A s well Ikaros-1 is expressed in L A K cells. However, what really stands out with the expression pattern shown is how faint bands appear in day 8. Not only does the phospho-image and gel picture show this (Fig 3.6a/b) but upon analysis of the data generated, day 8 shows the lowest expression level (Fig 3.6 Ikaros-1 52 Fig 3.6(a) 850 bases r Day 0 10 100 1000 r 10 Day 6 100 1000 r Day 8 Day 10 , i f l - ^ , inn , , 'ooo t r in inn Day 14 -4e-L A K 1000 + o o "H. o o a o a 3 Fig 3.6(b) 8 — - -— Fig 3.6 (a) is a phospho-image of PCR amplified Ikaros-1 cDNA. The size of the fragment is indicated on the left of the figure. Figure 3.6 (b) is a gel picture of the PCR amplified cDNA. The dark band adjacent to the * is the position of the 600 base band of a 100 base pair ladder that was run on either end of the gel. The first four wells of each day is PCR amplified c D N A from 10 cells. The next four wells is PCR amplified c D N A from 100 cells, and the last set of four wells from each day is PCR amplified c D N A from 1000 cells. The + indicates a positive control of either bone marrow (B), spleen (S) or thymus (T). The - is a negative control of H 2 0 . L A K indicated PCR amplified c D N A from 100 and 1000 L A K cells respectively. C0 O 0001-ISIIHB'SB 001 . >• • • mi Pill ISf OV Q iBII o 0001- -Hi 11 H sag™ IBM 001- g 01 0001- % illlii 001 Q 01. 0 0 0 1 . ^ 001- Q -lass 01-oooi. % CO 001- Q f ; llplt! » £>r 01-•"3" C O C M T - O s p u e g jo jaqiunN m 01 0001 001 01 0001 001 01 0001 001 01 i n <t co CM i - o (|Oj}uoo/a|dujes) /tysusjui pueg C/3 Cfl o .2 a g cd •a . 1 co O =3 XI &, co O XI & CD O o o o ctf CD ~ c o S O CD ^ T 3 « y or, O CO CO , s O -—' = 3 co o c . P e g 5b 5 ca w X co "S « Xi .5 N In ^ *3 <£ ill ait O X ?5 a 1 § .2 5 -a co 00. C C + H co Cu o c <? ?~> m O C3 fl - - H (D U CD x^  tfa ^ ^ CD w CO xi vo o H ^ £ vo td E 3 -3 d). This pattern suggests that expression of the gene is greatly reduced in the middle of the N K cell developmental pathway. The frequency of expression seems moderate as every aliquot of 1000 cells tested showed expression (Fig 3.6 c). Yet, the stepladder appearance of this figure illustrates that the frequency is not high. The less cells examined, the less expression is detected. While F ig 3.6 (c) day 0 shows frequency of expression to be high, F ig 3.6 (d) shows expression level to be somewhat high as well . The expression level appears to remain high then drop by day 8. Expression levels seem to be high again on day 10 and drop once again by day 14. L A K cells show an expression of Ikaros-1 that appears to be only slightly greater than what is shown for day 14 N K cells. While an on and off expression pattern of Ikaros-1 is evident from Fig 3.6 (d), the end resultant level of Ikaros-1 appears to be similar to that detected in N K cells. 3.6 ETS-1 There is a unique on and off pattern shown for Ets-1. Expression of the gene was detected day 0 and 6, then expression was off on day 8, on for day 10 and off again on day 14 (Fig 3.7 a). While not a lot of information can be gathered from the gel picture (Fig 3.7 b) the phospho-image illustrates an interesting pattern. N o expression on day 14 contradicts what the results of 100 and 1000 L A K cells indicate as normal for an N K cell, as expression is detectable in L A K cells (Fig 3.7 a). While on day 0 the frequency of expression seems to be moderate, by day 6 the frequency of expression is low, with only one cell sample showing expression out of all Fig 3.7 (a) r 10 991 bases Day 0 100 ETS-1 1000 r 55 Day 6 100 ^> r 1000 D a y 8 ^ r Day^lO 10 100 1000 10 100 1000 f v f \ f s / \ r s f I 9 Fig 3.7 (b) — 8 10 3.7 (a) is a phospho-image of PCR amplified Ikaros-1 cDNA. The size of the fragment is indicated on the left of the figure. Figure 3.7 (b) is a gel picture of the PCR amplified cDNA. The dark band adjacent to the * is the position of the 600 base band of a 100 base pair ladder that was run on either end of the gel. The first four wells of each day is PCR amplified c D N A from 10 cells. The next four wells is PCR amplified c D N A from 100 cells, and the last set of four wells from each day is PCR amplified c D N A from 1000 cells. The + indicates a positive control of either bone marrow (B), spleen (S) or thymus (T). The is a negative control of H 2 0 . L A K indicated PCR amplified c D N A from 100 and 1000 L A K cells respectively. oooi * 001 -J H 9S oooi. ^  OOL >• 01 Q O 0001- -001- $ 1 ' 1 01-oooi. % 1 1 - ' -001 Q 01-0001- ® r " CD 001. Q 01-oooi. ^  001. Q 01-0 0 CM T - O s p u e g jo jaqwriN CD • i — i 0001 001 01 0001 001 01 0001 001 01 0001 001 01 0001 001 01 « >n n N r . o © d d d d o (|OJVX)o/a|duies) A)!suayj| p u e g oo co co CO •g 'co o O H *c3 3 ime CO <u 6 0 ,3 CO o 3 co S CD ' *"H -4—* o cd CO o< CO O CD O phi o o o by Ti-T i ed r-i 3 *—I CO 3 o~ CU O -»-» CU T 3 O CO CO o CO CO -*-> r-on CO C T 1 6 0 'co PL, <+-! ds in cu* O ds in & & 3 B CO ca O ^> CO ,3 ca o "co CU CO N cu • » - H a 'H T i cu CO H—» .3 o w e of hei T 3 3 o o T i CO Xfl 3 res he ba exp -4—» >> HQ '55 o 3 >> o inte o 3 inte CU 0 ) cu ,3 +J CT1 cu H i -t 2 T ? ^-1 CU CO cu O H CO ed. by r~; CO t-i ed CO - P CO T 3 6 0 •> 3 T3 57 samples tested (Fig 3.7 c). There is no expression of Ets-1 on day 8. While expression occurs by day 10, the frequency of cells expressing Ets-1 on this day isn't high. Interestingly, no expression was detected on day 14. Attempting to quantify to some degree the levels of expression was difficult since the frequency of expression is not high. A s a consequence, the levels shown must be interpreted cautiously. For day 0, the frequency is relatively high (Fig 3.7 c), and the level of expression is relatively low (Fig 3.7 d). Day 6 shows one spike indicating high expression in one of the 1000 cell samples tested (Fig 3.7d), yet evaluating F ig 3.7 (c) reveals the frequency of expression is low and that would explain why high level of expression is seen in only one out of the four 1000 cell aliquots tested. Day 10 has a moderate level of expression, which is obviously higher than for day 8 (Fig 3.7 d). Lastly, it can be said that day 10 cell samples have a higher expression level than on day 14 as there was no detectable expression of Ets-1 on day 14. It is interesting to note that there is expression of Ets-1 in L A K cells but that there is no expression of Ets-1 in day 14 cells. Expression levels of Ets-1 in L A K cells appear low, yet it is a significant observation that the low level is still higher than what is seen for day 14. 3.7 OP-9 gene expression The phospho-image illustrates clearly that there are no bands detected for any of the genes of interest (Fig 3.8). This control clearly indicates that expression of the genes investigated is not due to contamination from the OP-9 stromal cell layer. The only bands apparent are for the positive control which is the G A P D H primers used on OP-9 c D N A . Fig 3.8 OP-9 cells 58 Ikaros-1 Id-2 ETS-1 CD CD CD CD CD CD G A P D H Notch-1 Jagged-1 H H & & H H H H cr a" d c. er s co co a - a" a" cr CD CD CD CD Fig 3.8 Phospho-image of c D N A derived from OP-9 cells. Tube 1 is a l x l O " 3 dilution and tube 2 is a l x l O " 4 dilution from the original tube of c D N A . P C R for each gene of interest was performed on c D N A for 25 cycles at annealing temperature of 58°C. Samples were run on a 30 m L 1% agarose gel for 1 hour. Alkal ine transfer was overnight, and P 3 2 labeled oligonucleotide probes were hybridized to the membrane at 58°C. 59 Chapter 4 Discussion 4.1 In Vitro Generation of N K - l i k e cells ES cells retain the ability to differentiate into all cell lineages in vitro. Before 1998, B and T cell progenitors were generated from ES cells in vitro, but N K cells were not. Nakayama and his group were the first to generate cytotoxic lymphocytes with characteristics of N K cells from ES cells (Nakayama et al. 1998). Our lab has taken this a step further by developing a culture system which produces N K - l i k e cells that exhibit a clearer N K cell phenotype by expressing the CD94 N K G 2 family of receptors. The Ly49 family of receptors have not been expressed on N K cells derived from ES cells to date. There are many factors to investigate to determine why the Ly49 family is not expressed on these N K like cells. It could be a factor of environment, it could be developmental gene expression levels or lack there of, or most likely it could be a combination of both factors. A n important aspect of understanding N K cell development lies in identifying the early progenitors that are committed to the N K lineage and determining patterns of gene expression in these cells. Investigating genes known to be involved in lymphoid cell development using an in vitro system is a solid first step to understanding the developmental pathway of an N K cell. Beginning with embryonic stem cells gives us the advantage of investigating the earliest stage possible. Our culture system allows us to look at gene expression at different stages of development and it allows us to compare gene expression in our in vitro cultured N K cell to a true N K cell. Once gene patterns can be elucidated, clues w i l l be apparent concerning how the N K cell develops and changes 60 can be made to the system in order to potentially induce Ly49 expression. Only when we have a complete N K cell expressing Ly49 receptors, we can begin to better understand the mechanism of receptor acquisition. 4.2 Ikaros Reflecting on what is shown in F ig 3.6 (d), it is not entirely surprising that expression levels of Ikaros-1 drop at day 8 and rise again at day 10. A n important cytokine change occurs on day 7 (Fig 4.1). Stem cell factor, 11-6,11-7 and Fit 3 ligand, which promote lymphoid cell development, are removed from the system and 11-2,11-15 and 11-18 are added (Fig 4.1). The latter cytokines are more specific in that they promote N K cell development. During the initial development of the N K cell (ES cell to putative lymphoid progenitor), Ikaros is expected to be expressed as it plays a large role in developing T-cells and B-cells (Sabbattini et al. 2001). The first 7 days of the system, beginning at CD34+ cells, is devoted to producing lymphoid progenitors. So it is not surprising that Ikaros is expressed at this stage. The sudden shift in cytokines may affect expression of other genes and promote the lymphoid progenitor to become an N K cell. Ikaros may not be needed during the critical change from lymphoid progenitor to N K cell. What is interesting to note however is that Ikaros expression is on and relatively high by day 10, and yet drops again by day 14. Expression on day 14 is quite similar in level to what is shown for L A K cells. This may indicate that Ikaros-1 is not involved in Ly49 receptor expression. The pattern of expression shown may be indicative of what happens in vivo in terms of Ikaros-1 expression in the developing N K cell. CM I •a o at at ta ml o in UJ 9 tea +p£aj /Qtea CO f-t H 1 0 CM i I joiiiidSojtl pioqduiiCi uouiuioa J i . —1 >o W J . #-M I E Li. 10 C/3 OO 19 0) vt o +-3E CU > o •5 CD > a o ,g ca •4—> 3 ca c a CD "8. £ o PH CD 6 o o a CD @3 C cd c o • I—I ca ca CD & X CD CD M » 3 ^ O CD fa +* PH O C+H T3 CD -i—> o CD -»—> CD •a CD O CD CD % CD C/3 oca ca CD CD o • 1—1 ca ca CD J-H X CD CD ca ca CD CD > x c CD o PS CD e CD ca GO £ CCJ CD &a C CD O • i—i G • l-H o 3 62 Knock out studies of Ikaros-1 results in lack of B , T and N K cells. It is thought that this protein is a transcriptional activator and repressor of gene expression (Cortes et al. 1999). Due to results shown in Fig 3.6 (d), it is reasonable to believe that Ikaros may affect gene expression during the early stages of N K cell development where lymphoid progenitors are developing. Other downstream signals may be needed for Ikaros to perform its function when lymphoid progenitors first differentiate to follow specifically an N K cell pathway. A s the cell commits to following this pathway, other signals are provided (by genes expressed as a result of new cytokines) and Ikaros appears to be needed again in the later stages of the N K cell development as expression is shown in L A K cells. 4.3 Ets-1 The transcription factor Ets-1 is essential to the differentiation of all lymphoid lineages. Knock-out studies have been the primary source of information regarding the roles of the Ets family of proteins. Specifically Ets-1 was found to be essential for B-cel l maturation, T-cell activation, and development and function of N K cells (Bartel et al. 2000). During morphogenesis, Ets-1 expression occurs among other places, in the thymus and spleen. Levels are low in the spleen the first week of the neonatal period, and levels increase in the spleen as maturation occurs (Maroulakou, Bowe 2000). Figure 3.7 (a/c) indicates that there is expression of Ets-1 during early development of the N K cell in vitro albeit at a low level (Fig 3.7 d). This corroborates what is reported in the literature. Since knock-out studies of Ets-1 result in no functional N K cells, it is logical to expect to see expression throughout development of the N K cell in vitro. So, it is puzzling to see no expression on days 8 and 14. It is however, not puzzling that expression is low on day 14, as expression levels are not high in spleen of the neonatal mouse, and our N K cells are similar phenotypically to fetal N K cells. Cytokines present in our culture system on day 0 and day 6 (where Ets-1 expression is seen) induce lymphoid cell differentiation from a totipotent ES cell (Fig 4.1). Day 8 shows no expression of Ets-1 (Fig 3.7 a). It is important to note that a cytokine change occurs on day 7, which promotes N K cell development (Fig 4.1). This shock to the system may affect the expression of Ets-1 as it is shown that expression is void on day 8 yet present again on day 10. A s explained for a similar result shown for Ikaros above, perhaps Ets-1 is not involved in the critical point in a N K cell developmental pathway where lymphoid progenitors commit to the N K cell pathway. Another possibility is that the cytokine change is too rapid and temporarily halts Ets-1 expression. It is also plausible that something may be missing from the in vitro system to maintain Ets-1 expression. In F ig 3.7 (d), Ets-1 expression in lacking in day 14 cells yet is present in L A K cells. This indicates Ets-1 may be involved in Ly49 generation and that its expression is not as high as it should be in vitro. The literature indicates that Ets-1 expression is seen in mature N K cells, which validate the expression present in L A K cells. Accordingly, expression should be seen in day 14 cells as well , and this is not the case. The hypothesis that something may be missing from the system is probable as Ets-1 expression is shown continually increasing in the spleen of a maturing mouse (Maroulakou, Bowe 2000). The literature implies expression is needed through out the development of the N K cell and not just for the first and last stages. 64 Ets-1 has been shown through many independent studies to be essential for N K cell development. Because it is not known at what point in the developmental pathway it functions, results seen in F ig 3.7 could be indicative of what occurs in vivo. Although, as explained above it seems more plausible that results from Fig 3.7 indicate there may be a deficiency in the in vitro system. Manipulation of the system resulting in constitutive expression of Ets-1 could present interesting results. It could potentially lead to an N K cell that expresses Ly49. Another thought promoting the idea that over-expression of Ets-1 in vitro may produce a N K cell with L y 49 stems from observation of in vivo expression patterns. Ets-1 is expressed in the thymus predominately neonatally. The bulk of inhibitory receptors found on neonatal N K cells are of the C D 9 4 / N K G 2 family, not of the Ly49 family. After birth, expression of the Ly49 family increases while that of C D 9 4 / N K G 2 decreases (Kubota et al. 1999). In vivo the thymus has Ets-1 expression and harbours a limited number of cells with Ly49. The spleen expresses Ets-1 and continues to expresses it in greater amounts as the mouse matures. In turn this maturing mouse generates N K cells with more Ly49 receptors. Perhaps Ets-1 is a factor which promotes Ly49 receptor acquisition. Our in vitro N K cells (day 14 cells), express C D 9 4 / N K G 2 , yet not Ly49 ((Lian et al. 2002)), which makes day 14 cells most similar to N K cells from an immature mouse. If Ets-1 could be expressed in vitro to a greater degree (to levels seen in the adult spleen), perhaps Ly49 expression could be induced. 65 4.4 Id-2 There is a growing body of evidence to suggest Id proteins play a role in the control of hematopoiesis and lymphopoiesis. Id-2 is a helix-loop-helix protein that sequesters and binds to basic helix-loop-helix (bHLH) transcription factors which renders them unable to bind D N A . The theory is that during differentiation, Id expression generally declines allowing for b H L H to homodimerize and trans-activate lineage specific genes. What is of specific interest is that Id-2 is the only member whose expression was maintained during ES cell derived hematopoietic differentiation (Nogueira et al. 2000). Results shown in F ig 4.1 are consistent with this in that Id-2 is expressed continually throughout development. It is expressed in C D 3 4 + cells and subsequently in all stages that follow. Maintaining expression of Id-2 through out the development of the N K cell is logical as Id-2 works by inhibiting E proteins involved in promoting T and B cell development. E l 2 and E47 are essential for B cell development. They work by controlling expression of P A X - 5 , which shuts off all developmental pathways of lymphoid precursors except that of the B cell pathway. E2 A proteins are involved in the commitment of T cell precursors to T cells. Id-2 blocks the action of these E proteins thereby encouraging N K cell development (Spits et al. 2000). Therefore it is of no surprise that Id-2 expression was apparent at each day of N K cell development tested (Fig 3.3 a/c, F ig 4.1). The specific mechanisms of how the Id2 gene is activated and how it participates in N K development remain unknown (Lian unpublished). It is easy to speculate that it interferes with other cell lineage pathways by blocking E proteins involved in T and B cell fate. 66 One study found that ld-2~'~ embryos had a marked reduction in N K cell numbers. Also it was found that there was no reduction in thymocytes. These results lead to the belief that Id-2 works at the level of the T / N K progenitor cell found in fetal thymus (Ikawa et al. 2001). A s it is, Id2 expression is not limited to fetal thymus progenitor cells. Id-2 is also found in progenitor cells in bone marrow (Rosmaraki et al. 2001). This implies it has a role in promoting N K cell development in bone marrow progenitors. It would be interesting to examine athymic mice that are Id-2~A. It would be reasonable to believe that there would be no N K cells. 4.5 Jagged-1 and Notch-1 The numerous studies that have implicated a role for Notch-1 in early hematopoietic cell fate make this large transmembrane receptor an interesting candidate to study in the developing N K cell. Albeit, studies have focused on its role in T cell development, but more pertinent to the objective of this thesis are the studies implicating its role in the T / N K cell precursor. This is of interest to us as we are investigating gene expression in the developing N K cell. If Notch-1 plays a role at such an early stage, then it inadvertently plays a role in N K cell development. In section 1.5 (d) it was explained how Notch-1 works via deltex signalling pathways. This results in lymphoid progenitors becoming T / N K progenitors (as Notch-1 represses B cell formation from lymphoid progenitors). More support for a role of Notch-1 at the lymphoid progenitor stage is from a study that looked at a constitutively active form of Notch-1. Mice receiving activated Notch 1-transduced B M contained immature CD4+ CD8+ T cells in the B M and exhibited a simultaneous block in early B cell 67 lymphopoiesis (Pui et al. 1999). This suggests Notch-1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions. It may act at a level of the common lymphoid progenitor influencing its decision to travel down the T / N K cell pathway. In our studies, we begin our system with an E S cell and we differentiate that cell into a lymphoid progenitor cell. We would expect to see Notch-1 expression in these early stage cells (and we do at a low level) as we are cultivating them with cytokines to eventually produce an N K cell (Fig 4.1). In doing so, we are promoting the development first of a lymphoid cell that can become an N K cell (hence the lymphoid progenitor we produce might be similar to the T / N K cell progenitor found in vivo). It appears from the phospho-image that days 6 and 10 have high expression relative to the other time points (Fig 3.5 a), but the analysis of expression level implies that on these days, Notch-1 has a very low frequency of expression (Fig 3.5 d). The low frequency of expression may be a result of the way in which Notch-1 expresses. It is thought that feedback through interaction with its ligand causes Notch-1 to express(Brenner 2000). Looking at our results for Jagged-1 (the ligand for Notch) we see that expression does not parallel that of Notch-1 (Fig 4.1). There is expression of Jagged-1 in C D 3 4 + cells, and this expression may initiate a higher than basal level of expression of Notch-1 in neighbouring ES cells. However, there is no expression of Jagged-1 throughout the development of the N K cell in vitro, and this may account for the low frequency of expression of Notch-1 seen in developing cells. It is worth noting that there is much higher expression in L A K cells of Notch-1 than in day 14 cells. Again, this implies that a normal level of Notch-1 expression is not achieved in our system. 68 It is also worth taking note of the fact that Jagged-1 is not expressed in the OP-9 stromal cell layer (Fig 3.8). In vivo Jagged-1 is expressed on bone-marrow stromal cells and thymic stromal cells (Osborne, Miele 1999). Again lack of expression of Jagged-1 is a probable reason for the abnormal expression level of Notch-1 seen in figure 3.8. For complete N K cell development, cytokines alone are insufficient to induce a mature N K phenotype. This suggests that additional factors or direct interaction with the bone marrow microenvironment may be required for final maturation (Lian unpublished). This is a reason why it is important to investigate OP-9 gene expression (Fig 3.8). There are studies focusing on proteins that may modulate Notch-1 thereby interfering with its binding affinity to certain ligands. One protein of particular interest is Fringe (Moloney 2000). Fringe is a glycosyltransferase which functions in the Golgi apparatus. It modulates carbohydrate chains on the Notch-1 receptor by adding sugar residues to the protein. A n important result of in vitro studies show Notch becomes more sensitive to signalling from the delta-like ligands, and less sensitive to signalling by the Jagged ligands (Fortini 2000). Assuming Jagged-1 is the appropriate ligand needed to push T / N K cell development, the presence of fringe on Notch-1 in our cultured cells could cause inhibition of ligand binding to Jagged-1 (present on other developing cells). This could negatively affect the push Notch-1 has to promote T / N K cell precursor. It would be interesting to investigate whether Fringe is expressed in our developing cells. Another study investigated Notch-1 ligands and suggest that it is Delta rather than Jagged-1 which when bound to Notch-1, promotes T / N K cell development (Jaleco 2001). If this is the case, then perhaps the results seen for Jagged-1 are not surprising (Fig 3.4). If Delta is the ligand which promotes T / N K cell development, then its expression levels would need to be investigated and compared with that of Notch-1. Altering its levels may alter Notch-1 levels and this may have an effect on the N K cell that develops from our culture system. 4.6 Stromal Environment A s it isn't a direct objective of this thesis, the stromal environment of the in vitro system wi l l not be discussed in depth. It is worth mentioning that the OP-9 stromal cell layer is an effective cell layer to promote differentiation of N K cells as it decreases the growth of macrophages, which tend to over populate the culture when other stromal layers are used. L y 4 9 + mature N K cells have been generated when hematopoietic progenitors were differentiated in the presence of OP9 stroma (Williams et al. 2000). Lymphotoxin (LT) a is a member of the T N F family and is secreted by activated lymphocytes. On developing N K progenitors, it appears to activate LT[3R + stromal cells and this provides the necessary microenvironment required for promoting N K development (Lian unpublished). This information shows the intimate nature of cell culture and culture stromal cells. Our N K like cells, which lack the Ly49 family of receptors, may benefit from some studies focused on L T production from the developing cells, and L T receptor expression on stromal cells. 4.7 Conclusion In vitro manipulation of ES cells to generate a culture system that functions to differentiate cells is useful as it provides a tool for investigating development. When I began my study, an in vitro culture system had been recently developed in my laboratory 70 in which embryonic stem cells are induced with various cytokines and growth factors to differentiate into N K cells. The objective of my work was to use this culture system to investigate the expression patterns of five genes which are known to play a role in lymphoid cell development based on gene knock-out studies. Id-2 seemed to be expressed consistently through out the system. Ikaros-1 and Ets-1 were interesting in that they showed an on/off expression pattern, Ets-1 with little expression on days 8 and 14, and Ikaros-1 with no expression on days 8 and 14. This implies that perhaps these transcription factors are not needed at the initial stage of N K cell differentiation from a common lymphoid precursor. Notch-1 and Jagged-1 were particularly interesting in that their expression patterns did not parallel each other. These results suggest that Jagged-1 may not be the ligand of choice for proper N K cell development as Notch levels were lower than expected. Ut i l iz ing this culture system by evaluating gene expression patterns provides insights into what genes are involved in development and to what degree. For example, the process of receptor acquisition is poorly understood, and this is partly due to the lack of understanding pertaining to the developmental pathway of N K cells. Understanding what genes are involved in the development of N K cells can elucidate clues pertaining to that developmental pathway. Understanding how an N K cell matures can shed light on mechanisms that initiate receptor expression. B y first gaining an understanding of the genes involved in N K cell differentiation we can eventually evaluate the role they play in N K cell development. Ultimately, experimentally manipulating gene expression within this in vitro system w i l l elucidate more clues in understanding the development of the N K cell Bibliography Bartel, F. O., Higuchi, T., and Spyropoulos, D . D. : Mouse models in the study of the Ets family of transcription factors. Oncogene 19: 6443-54, 2000 Barton, K . , Muthusamy, N . , Fischer, C , Ting, C. N . , Walunas, T. L . , Lanier, L . L . , and Leiden, J. M . : The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 9: 555-63, 1998 Biron, C. A . and Brossay, L . : N K cells and N K T cells in innate defense against viral infections. Curr Opin Immunol 13: 458-64, 2001 Brenner, M . : To be or notch to be. Nat Med 6: 1210-1, 2000 Carlyle, J. R., Michie, A . M . , Cho, S. K . , and Zuniga-Pflucker, J. C : Natural killer cell development and function precede alpha beta T cell differentiation in mouse fetal thymic ontogeny. J Immunol 160: 744-53, 1998 Carlyle, J. R., Michie , A . M . , Furlonger, C , Nakano, T., Lenardo, M . J., Paige, C. J., and Zuniga-Pflucker, J. C : Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes. J Exp Med 186: 173-82, 1997 Carretero, M . , Palmieri, G . , Llano, M . , Tull io, V . , Santoni, A . , Geraghty, D . E . , and Lopez-Botet, M . : Specific engagement of the C D 9 4 / N K G 2 - A killer inhibitory receptor by the H L A - E class lb molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated N K G 2 - A : evidence for receptor function in heterologous transfectants. Eur J Immunol 28: 1280-91, 1998 Carson, W . and Caligiuri, M . : Natural Ki l l e r Cel l Subsets and Development. Methods 9: 327-43, 1996 Chong, H . , Hutchinson, G . , Hart, I. R., and Vi l e , R. G . : Expression of B7 co-stimulatory molecules by B16 melanoma results in a natural killer cell-dependent local anti-tumour response, but induces T- cell-dependent systemic immunity only against B7-expressing tumours. Br J Cancer 78: 1043-50, 1998 Cortes, M . , Wong, E . , Koipally, J., and Georgopoulos, K . : Control of lymphocyte development by the Ikaros gene family. Curr Opin Immunol 11: 167-71, 1999 Deftos, M . and Bevan, M . : Notch Signaling in T cell development. Current Opinion in Immunology 12: 166-172, 2000 Diefenbach, A . , Jensen, E . R., Jamieson, A . M . , and Raulet, D . H . : Rae l and H60 ligands of the N K G 2 D receptor stimulate tumour immunity. Nature 413: 165-71, 2001 Dorfman, J. R. and Raulet, D . H . : Acquisition of Ly49 receptor expression by developing natural killer cells. J Exp Med 187: 609-18, 1998 Haire, R. N . , Miracle, A . L . , Rast, J. P., and Litman, G . W. : Members of the Ikaros gene family are present in early representative vertebrates. J Immunol 165: 306-12, 2000 Heemskerk, M . H . , B lom, B . , Nolan, G . , Stegmann, A . P., Bakker, A . Q., Weijer, K . , Res, P. C , and Spits, H . : Inhibition of T cell and promotion o f natural killer cell development by the dominant negative helix loop helix factor Id3. J Exp Med 186: 1597-602, 1997 Held, W. , Kunz, B . , Lowin-Kropf, B . , van de Wetering, M . , and Clevers, H . : Clonal acquisition of the L y 4 9 A N K cell receptor is dependent on the trans-acting factor T C F - 1 . Immunity 11: 433-42, 1999 73 Heusel, J. W. , Wesselschmidt, R. L . , Shresta, S., Russell, J. H . , and Ley, T. J.: Cytotoxic lymphocytes require granzyme B for the rapid induction of D N A fragmentation and apoptosis in allogeneic target cells. Cell 76: 977-87, 1994 Ikawa, T., Fujimoto, S., Kawamoto, FL, Katsura, Y . , and Yokota, Y . : Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2. Proc Natl Acad Sci US A 98: 5164-9, 2001 Keller, G . M . : In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 7: 862-9, 1995 K i m , S., Iizuka, K . , Agui la , H . L . , Weissman, I. L . , and Yokoyama, W . M . : In vivo natural killer cell activities revealed by natural killer cell- deficient mice. Proc Natl Acad Sci USA 97: 2731-6, 2000 Kubota, A . , Kubota, S., Lohwasser, S., Mager, D . L . , and Takei, F.: Diversity of N K cell receptor repertoire in adult and neonatal mice. J Immunol 163: 212-6, 1999 Lanier, L . L . : N K cell receptors. Annu Rev Immunol 16: 359-93, 1998 Lanier, L . L . : The origen and function of natural killer cells. Clinical Immunology 95: S14-S18, 2000 Lanier, L . L . : On guard—activating N K cell receptors. Nat Immunol 2: 23-7, 2001 Lanier, L . L . , Ruitenberg, J. J., and Phillips, J. FL: Functional and biochemical analysis o f CD16 antigen on natural killer cells and granulocytes. J Immunol 141: 3478-85, 1988 Leibson, P. J.: Signal transduction during natural killer cell activation: inside the mind o f a killer. Immunity 6: 655-61, 1997 L i , R., Pei, H . , and Watson, D . K . : Regulation of Ets function by protein - protein interactions. Oncogene 19: 6514-23, 2000 Lian, R. H . , Maeda, M . , Lohwasser, S., Delcommenne, M . , Nakano, T., Vance, R. E . , Raulet, D . H . , and Takei, F.: Orderly and Non-Stochastic Aquisition of C D 9 4 / N K G 2 Receptors by Developing N K Cells Derived from Embryonic Stem Cells In Vitro. J. Immunology, 2002 Ljunggren, H . G . and Karre, K . : In search of the 'missing self: M H C molecules and N K cell recognition. Immunol Today 11: 237-44, 1990 Lodolce, J. P., Boone, D . L . , Chai, S., Swain, R. E . , Dassopoulos, T., Trettin, S., and M a , A . : IL-15 receptor maintains lymphoid homoestasis by supporting lyphocyte homing and proliferation. Immunity 9: 669-676, 1998 Maroulakou, I. G . and Bowe, D . B . : Expression and function of Ets transcription factors in mammalian development: a regulatory network. Oncogene 19: 6432-42, 2000 Mason, L . H . , Gosselin, P., Anderson, S. K . , Fogler, W . E. , Ortaldo, J. R., and McVica r , D . W. : Differential tyrosine phosphorylation of inhibitory versus activating Ly-49 receptor proteins and their recruitment of SHP-1 phosphatase. J Immunol 159: 4187-96, 1997 Mavrothalassitis, G . and Ghysdael, J.: Proteins of the ETS family with transcriptional repressor activity. Oncogene 19: 6524-32, 2000 McVica r , D . W . and Burshtyn, D . N . : Intracellular signaling by the killer immunoglobulin-like receptors and ly49. Sci STKE 2001: R E 1 , 2001 Mi l le r , J. S.: The biology of natural killer cells in cancer, infection, and pregnancy. Exp Hematol29: 1157-68, 2001 Nakamura, M . C , Linnemeyer, P. A . , Niemi , E . C , Mason, L . H . , Ortaldo, J. R., Ryan, J. C , and Seaman, W . E. : Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity. J Exp Med 189: 493-500, 1999 Nakayama, N . , Fang, I., and Elliott, G . : Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence o f vascular endothelial growth factor. Blood 91: 2283-95, 1998 Nogueira, M . M . , Mitjavila-Garcia, M . T., Le Pesteur, F., Fi l ippi , M . D . , Vainchenker, W. , Dubart Kupperschmitt, A . , and Sainteny, F.: Regulation of Id gene expression during embryonic stem cell-derived hematopoietic differentiation. Biochem Biophys Res Commun 276: 803-12, 2000 O'Callaghan, C. A . : Molecular basis o f human natural killer cell recognition o f H L A - E (human leucocyte antigen-E) and its relevance to clearance o f pathogen- infected and tumour cells. Clin Sci (Lond) 99: 9-17, 2000 Osborne, B . and Miele , L . : Notch and the immune system. Immunity 11: 653-63, 1999 Payne, K . J . , Nicolas, J. FL, Zhu, J. Y . , Barsky, L . W. , and Crooks, G . M . : Cutting edge: predominant expression of a novel Ikaros isoform in normal human hemopoiesis. J Immunol 167: 1867-70, 2001 Pui , J. C , Al lman, D . , X u , L . , DeRocco, S., Karnell, F. G. , Bakkour, S., Lee, J. Y . , Kadesch, T., Hardy, R. R., Aster, J. C , and Pear, W . S.: Notch l expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11: 299-308, 1999 Rosmaraki, E . E . , Douagi, I., Roth, C , Colucci , F. , Cumano, A . , and D i Santo, J. P.: Identification of committed N K cell progenitors in adult murine bone marrow. Eur J Immunol 31: 1900-9, 2001 Roth, C , Carlyle, J. R., Takizawa, H . , and Raulet, D . H . : Clonal acquisition of inhibitory Ly49 receptors on developing N K cells is successively restricted and regulated by stromal class I M H C . Immunity 13: 143-53, 2000 Sabbattini, P., Lundgren, M . , Georgiou, A . , Chow, C , Warnes, G . , and Di l lon , N . : Binding o f Ikaros to the lambda5 promoter silences transcription through a mechanism that does not require heterochromatin formation. EmboJ 20: 2812-22, 2001 Schmitt, R. M . , Bruyns, E. , and Snodgrass, H . R.: Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev 5: 728-40, 1991 Sementchenko, V . I. and Watson, D . K . : Ets target genes: past, present and future. Oncogene 19: 6533-48, 2000 Snodgrass, H . R., Schmitt, R. M . , and Bruyns, E . : Embryonic stem cells and in vitro hematopoiesis. J Cell Biochem 49: 225-30, 1992 Spits, H . , Couwenberg, F., Bakker, A . Q., Weijer, K . , and Uittenbogaart, C. H . : Id2 and Id3 inhibit development of CD34(+) stem cells into predendritic cell (pre-DC)2 but not into p r e - D C l . Evidence for a lymphoid origin o f pre-DC2. JExp Med 792: 1775-84,2000 Toor, A . A . , Lund, T. C , and Mil ler , J. S.: T-cell factor-1 expression during human natural killer cell development and in circulating CD56(+) bright natural killer cells. Exp Hematol 29: 499-506, 2001 Toyama-Sorimachi, N . , Taguchi, Y . , Yagita, H . , Kitamura, F. , Kawasaki, A . , Koyasu, S., and Karasuyama, H . : Mouse CD94 participates in Qa-1 -mediated self recognition by N K cells and delivers inhibitory signals independent of Ly-49. J Immunol 166: 3771-9, 2001 Trinh, L . A . , Ferrini, R., Cobb, B . S., Weinmann, A . S., Hahm, K . , Ernst, P., Garraway, I. P., Merkenschlager, M . , and Smale, S. T.: Down-regulation of T D T transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator. Genes Dev 15: 1817-32, 2001 Trowsdale, J.: Genomic structure and function in the M H C . Trends Genet 9: 117-22, 1993 Vales-Gomez, M . , Reyburn, H . , and Strominger, J.: Interaction between the human N K receptors and their ligands. Crit Rev Immunol 20: 223-44, 2000 Westgaard, I. H . , Berg, S. F. , and Orstavik, S.: Identification of a human member o f the Ly-49 multigene family. Eur J Immunol 28: 1839, 1998 Wil l iams, N . S., K lem, J., Puzanov, I. J., Sivakumar, P. V . , Bennett, M . , and Kumar, V . : Differentiation of NK1.1+, Ly49+ N K cells from flt3+ multipotent marrow progenitor cells. J Immunol 163: 2648-56, 1999 Wil l iams, N . S., Kubota, A . , Bennett, M . , Kumar, V . , and Takei, F. : Clonal analysis o f N K cell development from bone marrow progenitors in vitro: orderly acquisition o f receptor gene expression. Eur J Immunol 30: 2074-82, 2000 Yokota, Y . , Mansouri, A . , M o r i , S., Sugawara, S., Adachi, S., Nishikawa, S., and Gruss, P.: Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397: 702-6, 1999 Yokoyama, W . M . : Natural killer cell receptors. Curr Opin Immunol 10: 298-305, 1998 Yokoyama, W . M . : Now you see it, now you don't! Nat Immunol 1: 95-7, 2000 Young, J. D . and Cohn, Z . A . : Cellular and humoral mechanisms of cytotoxicity: structural and functional analogies. Adv Immunol 41: 269-332, 79 APPENDIX A 80 I d - 2 cDNA gaaccgagcc tggtgccgcg cagtcagctc agccccctgt ggcggctccc t c c c g g t c t c t c c t c c t a c g agcagcatga aagccttcag gtccggtgag tccgttagga aaaacagcct gtcggaccac agcttgggca tctcccggag caaaaccccg gtggacgacc cgatgagtct gctctacaac atgaacgact g c t a c t c c a a gctcaaggaa ctggtgccca gcatccccca gaacaagaag gtgaccaaga tggaaatcct gcagcacgtc at.cgat.taca t c t t g g a c c t gcagatcgcc ctggactcgc a t c c c a c t a t cgtcagcctg catcaccaga gacctggaca gaaccaggcg tccaggacgc gcctgaccac cctgaacacg gacatcagca t c c t g t c c t t gcaggcatct g a a t t c c c t t c t g a g c t t a t gtcgaatgat agcaaagtac t c t g t g g c t a aataaatggc atttggggac t t t t t c t t t t c t t t t t a c t t t c t c t t t t t c t t t t g c a c a a gaagaagtct acaagatctt t t a a g a c t t t t g t t a t c a g c c a t t t c a c c a ggagaacacg ttgaatggac c t t t t t a a a a agaaagcgga aggaaaacta aggatgatcg t c t t g c c c a g g t g t c g t t c t ccggcctgga ctgtgatacc g t t a t t t a t g agagactttc a g t g c c c t t t ctacagttgg a a g g t t t t c t t t a t a t a c t a t t c c c a c c a t ggggagcgaa aaggttaaaa aaaagaaaaa aatcacaagg aattgcccaa tgtaagcaga c t t t g c c t t t tcacaaaggt ggagcgtgaa ttccagaagg acccagtatt cggttactta aatgaagtct tcggtcesgaa a t g g c c t t t t tgacacgagc c t a c t g a a t g c t g t g t a t a t a t t t a t a t a t a a a t a t a t a t a t a t t g a g t g a a c c t t g t g g a c t c t t t a a t t a g a g t t t t c t t g t a t a g t g gcagaaataa c c t a t t t c t g cattaaaatg taatgacgta c t t a t g c t a a a c t t t t t a t a aaagtttagt t g t a a a c t t a a c c c t t t t a t acaaaataaa t c a a g t g t g t t t a t t g a a t g t t g a t t g c t t g c t t t a t t t c agacaaccag t g c t t t g a t t t t t t t t a t g c t a t g t t a t a a ctgaacccaa ataaatacca g t t c a a a t t t atgtagactg t a t t a a g a t t ataataaaat g t g t c t g a c a tcaaaaaaaa aaaaaa Id-2 C D N A from NCBI accession number m69293. Shaded underlined sequence is that used to design PCR primers for Id-2. Region amplified is 733 bases. Bold italic sequence was used to design oligonucleotide probe. Ikaros-1 cDNA ccttggggag gcacaagtct gttgataacc tgaagacaat ggatgtcgat gagggtcaag ac a t g t c c c a agtttcagga aaggagagcc ccccagtcag tgacactcca gatgaagggg atgagcccat g c c t g t c c c t gaggacctgt c c a c t a c c t c tggagcacag cagaactcca agagtgatcg aggcatgggt gaacggcctt tccagtgcaa ccagtgtggg g c c t c c t t t a cccagaaagg c a a c c t c c t g cggcacatca agctgcactc gggtgagaag c c c t t c a a a t g c c a t c t t t g caactatgcc tgccgccgga gggacgccct caccggccac ctgaggacgc a c t c c g t t g g taagcctcac aaatgtggat attgtggccg gagctataaa cagcgaagct ctttagagga gcataaagag cgatgccaca actacttgga aagcatgggc cttccgggcg tgtgcccagt cattaaggaa gaaactaacc acaacgagat ggcagaagac ctgtgcaaga taggagcaga gaggtccctt gtcctggaca ggctggcaag caatgtcgcc aaacgtaaga g c t c t a t g c c t c a g a a a t t t cttggagaca agtgcctgtc agacatgccc tatgacagtg ccaactatga gaaggaggat atgatgacat cccacgtgat ggaccaggcc atcaacaatg ccatcaacta cctgggggct gagtccctgc gcccattggt gcagacaccc cccggtagct ccgaggtggt gccagtcatc agctccatgt accagctgca caagcccccc tcagatggcc ccccacggtc caaccattca gcacaggacg ccgtggataa c t t g c t g c t g ctgtccaagg c c a a g t c t g t gtcatcggag cgagaggcct ccccgagcaa cagctgccaa gactccacag atacagagag caacgcggag gaacagcgca gcggccttat ctacctaacc aaccacatca acccgcatgc acgcaatggg c t g g c t c t c a aggaggagca gcgcgcctac gaggtgctga gggcggcctc agagaactcg caggatgcct t c c g t g t g g t cagcacgagt ggcgagcagc tgaaggtgta caagtgcgaa cactgccgcg t g c t c t t c c t ggatcacgtc a t g t a t a c c a ttcacatggg ctgccatggc tgccatggct ttcgggatcc c t t t g a g t g t aacatgtgtg g t t a t c a c a g ccaggacagg t a c g a g t t c t c a t c c c a t a t cacgcggggg gagcatcgtt accacctgag ctaaacccag ccaggcccca ctgaagcaca aagHag'ctg gttatgcctc c t t c c c g g c a gctggaccca cagcggacaa tgtgggagtg gatttgcagg c a g c a t t t g t t c t t t t a t g t t g g t t g t t t g g c g t t t g a t t tgcgttggaa g a t a a g t t t t t a a t g t t a g t gacaggattg c a t t g c a t c a ggaacattca caacatccat c c t t c t a g c c Ikaros-1 C D N A from NCBI accession number L03547. Shaded underlined sequence is that used to design PCR primers for Ikaros-1. Region amplified is 850 bases. Bold italic sequence was used to design oligonucleotide probe. 81 Ets-1 cDNA a t g t t t t a t t c t t t g t a g a a a c t t g a c t t c c t t a a t a a t a t c c t g c c a t t t c t cag taga c t g c t t t c c a c ta taagaca t t t t c t t g a t ggccaccact t c a t t a g t a c gcaca tc t cg tgcaaaata t a c tgc t ccaa a g c t c c c a t g g c c g a t t t c t c tgaga tc tg tgaagagct t t t c c g g a t c t t c t t t g a a g a agagctc tgc aa t t t caggg t c c t t t a g a c t g c c t g c t c t g t t a g t t g a t ggcatcc tgc tgagcagtca g c t g t c t g a t t c t a a t t t t a t a c t g c t a t c c c a a t g c t t g g t t c a t c t g t atgaatgaag ccacgctggc a t a a a a t t t c gtaacaggaa a t c t t g a a t a t a t t t t t t a a aaatccagcc atgaacaagt tcagaaacca agt tgcaggc cagccagaga ac t cac t cag cactgaatac acagta tagt gagaagcaaa c t t a c t c t c a tcccgccggc c c c c t c a a c t ccgggcacca tgaaggcggc cg t cga t c t c aagccgactc t c a c c a t c a t caagacagaa aaagtggatc t c g a g c t t t t c c c t t c c c c g gacatggaat g tgcagatgt c c c g c t g t t a ac tccgagca gcaaagaaat ga tg tcccaa gcct tgaaag c t a c t t t c a g t g g t t t c a c a aaagaacagc agcgactggg aa tccccaaa gacccccggc agtggacaga aacccacgtc cgggactgggN>'t'g'atgtgggc tgtgaat 'gag jEtcagcctga aaggtgtgga c t t ccagaag t t c t g c a t g a gtggagcagc ac tg tg tgcc ctgggtaaag a a t g c t t c c t cgagctggct c c a g a c t t t g t tggggata t cctgtgggag ca tc tagaga tcc tgcagaa agaggatgtg aaaccata tc aggt taatgg agccaaccct acc tacccag a a t c c t g t t a cacc tcgga t t a c t t c a t c a gc tacgg ta t cgagcatgct c a g t g t g t t c c t c c c t c a g a g t t c t cagag c c c a g c t t c a tcacagagtc c ta tcagacg c t g c a t c c t a tcagctcgga agaactcctg- tccctcaagt atgagaacga c t a c c c t t c t g t c a t t c t c c aggaccc tc t ccagacagac acc t tgcaga cagac t ac t t tgcca tcaag caagaggtgt t aac tccaga caacatgtgc ctggggagag ccagtcgtgg taaactcggg ggccaggact c t t t t g a g a g cgtagagagc t acga tag t t g tgaccgcct cacccagtcc tggagcagcc a g t c a t c c t t caacagcctg cagcgggtcc c c t c c t a t g a cagct tcgac tacgaggat t a t cccgc tgc cc tgcccaac cacaagccca agggcacct t caaggactat gtgcgtgacc g tgc tgacc t caacaaggac aagcc tg tca t t c c t g c t g c t gccc tggc t ggctacacag gaagtgggcc ga tccagctg t g g c a g t t t c t t c t g g a a t t a c t c a c t g a t a a g t c t t g t c a g t c c t t t a t cagctggaca ggagatggct gggaat tcaa g c t t t c t g a c ccagatgagg tggccaggag atggggaaag aggaaaaaca aacctaagat gaa t ta tgag aaactgagcc g t g g c c t t c g c t a c t a t t a t gacaaaaata t ca t ccacaa gacggcgggc aagcgctacg t a t a c c g c t t tg tg tgcgac ctgcagagcc tgc tgggata cacccc tgaa gagctgcacg cca tgc tgga tg taaagccg ga tgc tgac t agtcatggac agacgcgcag aaggaagggg ctgggggaac c tgctgagac c t t t c a a g a g c t taagacga t a Ets-1 CDNA from NCBI accession number x53953. Shaded underlined sequence is that used to design PCR primers for Ets-1. Region amplified is 991 bases. Bold italic sequence was used to design oligonucleotide probe. Notch-1 cDNA gtgg tg tgcg t caacg tccg a tccccgccg gccaccccaa gaggccgccg ccgggctgcg ggcagctggc gagcaggcat gccacggctc c tgacgcccc t t c t c t g c c t aacgc tgc tg cccgcgcgcg ccgcaagagg c t tgaga tgc tcccagccaa gtgggacctg cctgaatgga ggtaggtgcg aagtggccag cggcactgaa g c c t g t g t t g ccagcggcag c t t t g tgggc caacgatgcc aggaccccaa t c c t t g c c t c agcacacggt gtaagaatgc tggaacgtgc t a t g t t g t g g accatggtgg catcgtggac t a t g c c t g c a g c t g t c c c c t g g g t t t c t c t gggcccc tc t gcctgacacc tc tggacaag ccc tgcc tgg ccaacccc tg ccgcaatggg ggcacc tg tg acc tgc t cac tc tcacagag tacaagtgcc g c t g c t c t c c agggtggtca ggaaaatcat gtcagcaggc t g a c c c c t g t gcc tccaacc cc tg tgccaa tggtggccag t g c c t g c c c t t t g a g t c t t c a t a c a t c t g t cgctgcccgc c t g g c t t c c a tggccccacc tgcaggcaag a t g t t a a t ga gtgcagccag aaccctgggc tg tgccgcca tggaggccac tgccacaatg agatcggctc c t a t c g c t g t g c c t g c t g t g ccaccca tac tgg tccccac tg tgaac tgc cc t a tg tgcc c tgcagcccc t c accc tgcc agaatggagc aacc tgccg t cctacagggg acaccaccca cgagtgtgcc t gc t t gccag g t t t t g c t g g acagaactgt gaagaaaatg tgga tgac tg tccaggaaac aactgcaaga atgggggtgc c tg tg tggac ggcgtgaata c c t a c a a t t g ccgc tgccca ccggaggtga cgggtcagta c tgtacagag gatgtggacg aa tg tcagc t ca tgccaaa t gcctgccaga atgcgggaac ctgccacaac acacacggcg gc tacaac tg t g t g t g t g t c aatgggtgga ctggcgagga ctgcagtgag aaca t tga tg ac tg tgccag t gccgcc tg t t t c caggg tg c c a c t t g c c a cgaccgtg tg g c t t c c t t c t actgcgaatg tccgcatggg cgcacaggtc t g c t g t g c c a cc tcaagca t gcg tgca tca gcaacccc tg caacgagggc t c c a a c t g t g acaccaaccc tgtcaacggc aaacgaatc t gcacc tgccc ctcggggtac acagggccag cc tgcagcca ggacgtggat gagtgtgatc tgggtgccaa ccg t tg tgag cacgcaggca aa tgcc tcaa cacactgggt t c t t t t g a g t gccag tg t c t acagggctac acgggacccg gctgtgagat t g a t g t t a a t gag tgca tc t ccaaccca tg tcagaatgac gccac t t gcc tggaccagat tggggagttc caa tgca ta t g ta tgccagg t t a tgaaggt g t a t a c t g t g 82 aaatcaacac gttccaatgt acaccatgca acacagggac ggatggtgtg aatgagtgcc tatgcctcaa ctctggcacc tgtaacgtca cgggcttcac cagtaacccc tggagtggaa gcaaggacat catcaacgaa tgcaactgtc gcaaaaacag gcaaggtcaa cagaacacca tcgatgactg cgactgcctg caaaatggag tccactgcga tatcaactcc tgtgattcac cacagggcta tggcaggtgc gacgtgctca atggagggct ctgtgaggac ggcggctttt tgtcccagcc ggggacacag agccccaagt gcttcgtcgg ccagaactgt gagtcagtca ccgcccgtgg ttgtggcagc ctgggatcct acaatcaggg cgggctactg attgaggagg ataatcacgc gcagtctcta c t c t t t g a t g accacttcag tgctgagcat ctacggaaca gtgatgcgca caagcgctct gagctggacc catcctcgca c c t c a a t a t t cacctcatgt gcaagcgccg gaagcggaga ctgatggacg cagtagttct tgacctgcgc aatgttcgag gcaacagtga caaccagaca ggatgagtgc cagtgcccca agaacggtgc ccactgcgaa g c c a c c t t t a acagccaacc gggaaccaca tgtctggaca acattgacga ttgccgctgc tgcatccacg caaactgtga gaccagtggc tgtgcctcca ctctgccata cggcgtatgc acctgcgagg atggcagcta ccgccccaac cccggcttcc ccaattgcac gaacaacaca t t c a c c t g t c ggccctgtct cactggtctc tggcagacca gtgtgtcctg ctgtgtggat gaggtggacg cctgcaagtg ctgccagaat ggtgtacact gcttcaacaa ggagcggtgt gtgcagcgtg tcaatggctg a t t c a t c t g t ttacgctgtc tcaccggccc cacctgtgag tgccacatcc cctgtgagct atgtggctgg cagtgctgga gcttcgactg tgatggccac gtacccgagc a c t c c t t c c a aggccagcag acagtgggtt ccatggacat gtgcttccag ccttacaaga acgtggcagc gcgccagcat gagcccctcg acaatcagaa ccctgacctg atgtctgcca gaccagatgg agaagaagaa gaccgcaccg gccagcagcc aaggcttcaa caagtgcctg gtggacattg cctgcctgtg gtgccgccat gggcccaact agattgatgg atgtgcgggc cccgagggct gagcttgccg catcaacaac tacgtatgca acccctgcct tacaggagcc aaggagtctg ttgacatcaa ccgctgcctc ccgtgtcaca agggtgcctt tgactgtgtg cctgactgta tgtgtccacc gcacggtggt aactgccaga acacgcagta tgaggtggct gagggagata agtgctcacc tgtggctggc gggggtacct gtgagatcaa tggcacctgt gagggtgatg tt a a t g a c t t caggggcaaa aggtgccctg tcaacggtgg tgagtgccag cccacatccg tggactacag gcctgagtgc gatggtggcg a g t a t t t t a g ccagctcacc tgcgaccagg ggctggcagc ct t t c t g c g g a t g a t c t t c c gggccacctc ccgtggctcc agtgccaccg ttgaggccgt ggccgccttc ggccagctct gcgaggactc cgagtgggga agtgatcaga tggccccaac cttcacaccc gatgcacctg gggagaccgc cctgtctgca cgggcacctg gatgggccca acgagtgtga ccagccaggc gggggcacct gtgagatcaa ctacgaatgt agcccctgcc accatgaccc ggatggcctc aacgagtgtg cctgccgaga gaaccagggg acgtgtgagg aagactatga tgagtgtgtg tgccaggccg atgggggttc ctgtgaggag gacagctaca ctgagagctc tggcttcacg acctgccaag accttgtgcg ccactgtgag gcacagaagc aacattactg taacccctgc taccatgggt gcattgatct tgttgatgac gtggaccagg tcaatgaatg ccactgcgag ccttgcaaga cgggcttcga tacatgcatc ttcccagcca agaacccttt cttcacaggt caggtggatg actgctccct cgacggccac gagggacagt gctgtaacag gggcaccctg gagctcagcc cgtactatgg t t c a c t g c t t a t t g t c t a c c atgtggctgc gaagagtgag gtgctcctgt ggttccctga agtcggcctc gacgaagacc ctgaccacag accgcctcag ctcatgattg c t g t c a t c t c c t t g c a c t t g caatggccac tgccagtatg acacctatac ccctgacccc tacacaggcc gccaggaccg cctggatgac gcctgtgaac acaacggggg cacgtgcctg aatgggtaca agtccaaccc aggcttcagt acctgcattg tggtgttggc gagtttctcc aaaagcccat gctatacagg ctgcaccgat gacatcaatg catgtacctg ct g c t t c a a t ggcagctact acagctatgg ctggtgcgac tgccgcagcg gaggcattga ccactgccag cagaatggag ctaactgctc gaccaactcc tgccatcccc tgggtggcta tctctccaac tgccgggctg atgggggtgt gggtgccaca tcgggcccac gcagcccctg ctaccgctgt ggcgctggcc caggcaataa caacttcaat tgtgacagcc gcaaccccct tgccgaatgt gtcctggtgg acgtgctgca ccacgaggaa cctggtacca tggagatcga cttcctaggt ccggtggagc t c t t t g t g g g gggtttcaaa aagcccctga tggagaccaa gcagtggacc ggggaggtgg cctcctgcag tgacttcatc gctgcccgat tgcatggaca atgtggatga ctgcgtgtgt tgccactatg atcactgtga tgacaactcc tgcgccagca caggctacac cacttgtgag tccgaggtca agtgtgactg ttgtgtcaac ggccctaatt atgatgtcgc cccatgtgct t g t g t c t g t c gtcgccatgg tcgcaactgt ggcatcaaca aatgtgccag ccccgtgggc ggtggtacct gtcagtatga tacttataag tcggctccct gctggactgg c g t c a c t c t c l gcaggctaca ctacctgcac cgaggagatc tacaagtgtt cccttgaccc tacctgcacc ccctgtgacc gccacactgg ctgtgccgtg tgtgagaatg gtagtcccac tgtgggtagc ctatgccctg cggacattcc ggtctgcaac gacccctgga agtgcaactc gtatgaccag gagtgggatg tg c t g c t t c c caccaacgtg gagctgcgca gtggtgggcg caaccggcaa gctcttgcgt ctccgctgcc ctgtggggtg gtgtcagagg agaatgcctc gaagttccgg cagcagcacc atgctgactg tggagggggc taccagggcg a c t c t c g t t c agatccatga gtgtgccagc acagaaggtt g t t c c t g t a a gaccaacatc t a c c t c t g c t acccctgtga aggaagcatg gatggcatcg acgagtgcaa tgcccctggg ggtggcacct gccagaccaa tggatacaag accagcccct ccacaggctg ggcctcctgc gagagtgaca cagccttctg caatccctgc ttcaatggca gtgtggatgg tgtcaatgag tgtacctgcc gcaagaatgg cgtcaactgc ctgtgccagc cgggcagcta t g a c t a t c t c aacgagtgcc cctgcccccg tgcctcccga tgcccaccag cacgtggcac acgccgctgt gcctccaaca atgcccgcac ctgcctatgc aacccctgct ccaaattcaa cccaccgcag ctgcagtgta agaactgcac ggccggctgc tactgcaagg gcctagactg acccgaccag gtcttcaagc agcacccaat ccagcgcagg tgtgtgcagt cacttggcag ctcgcagctg ctgctgtccc ccagcaagaa agatggtgct tttgaggagc tggacgctgc catggatgtc cttgagacag ccagcttgca agatcgtcga 83 aagcgccttg aggccagtgc agatgccaac atccaggaca acatgggccg t a c t c c g t t a catgcagcag tttc t g c a g a tgctcagggt gtcttccaga tcctgctccg gaacagggcc acagatctgg atgcccgaat gcatgatggc acaactccac tgatcctggc tgcgcgcctg gccgtggagg gcatgctgga ggacctcatc aactcacatg ctgacgtcaa tgccgtggat gacctaggca agtcggcttt gcattgggcg gccgcggtga acaatgtgga t g c t g c t g t t gtgctcctga agaacggagc caacaaggac atcgagaaca acaaggagga gac t t c c c t g t t c c t g t c g a tccgccgtga gagctatgag actgccaaag tgttgctgga ccactttgcc aaccgggaca tcacggatca catggaccga ttgccgcggg acatcgcaca ggagcgtatg caccacgata tcgtgcggct tttggatgag tacaacctgg tgcggtcccc acagctgcat ggcactgccc tgggtggcac acccactctg tctcccacac tctgctcgcc aaatggctac cctggcaatc tcaagtctgc cacacagggc aagaaggccc gcaagccaag caccaaaggg ctggcttgtg gtagcaagga agctaaggac ctcaaggcac ggaggaagag ttcccaggat ggcaagggct ggctgttgga cagctcgtcg agcatgctgt cgcctgtgga ctccctcgag tcaccccatg g c t a c t t g t c agatgtggcc tcgcaccccc t c c t c c c c t c cccattccag c a g t c t c catACCa'tgc'cfcctJScagccacctg cctggtatgc ctgacaccca cctgggcatc agccacttga atgtggcagc caagcctgag atggcagcac tggctggagg tagccggttg gcctttgagc accccccgcc acgcctctcc cacctgcctg tagcctccag tgcctgcaca gtgctgagta ccaatggcac cggggctatg aatttcaccg tgggtgcacc ggcaagcttg aatggccagt gtgagtggct tccccggctc cagaatggca tggtgcccag ccagtacaac ccactacggc cgggtgtgac gccgggcaca ctgagcacac aggcagctgg gctccagcat agcatgatgg ggccactaca cagcagcctc tccaccaata c c t t g t c c c c g a t t a t t t a c cagggcctgc ccaacacacg gctggcaaca cagcctcacc tggtgcagac ccagcaggtg cagccacaga acttaccact ccagccacag aacttacagc caccatcaca gccacacctc agtgtgagct cggcagccaa tgggcacctg gggcggagct tcttgagtgg ggagcccagt caggcagalTgllSaliaaccgct' gggccccagc agtctgcctg tgcacaccat tctgccccag gaaagccagg ccctaccaac atcactgcca tcctccatgg tcccacccat gaccactacc cagttcctga cccctccatc acagcacagt t a c t c c t c c t cccctgtgga caacaccccc agccaccagc tgcaggtgcc agagcccact t t c c t c a c c c catcccctga gtcccctgac cagtggtcca gctcctcccc gcattccaac a t c t c t g a t t ggtccgaggg catctccagc ccgcccacca atgtgggatg caggacccca g c t t c c g t t c ccaagccctg ttggaagtcc tttccagtgc ttcaggatgc tggggcgacc aaaggagctt tttaaaaaat g t t t t t a t a c aaaataagag gacaagaatt t c a t t t t t t t t t t t a g t a t t t a t t t a t g t a c t t t t a t t t t ccacagaaac a c t g c c t t t t t a t t t a t a t g t a t t g t t t t c tatggcacta ggggaaaaac a t a t c t g t t c caagaaaata aactagttct cagagccttg a t t t t c c t g g tcagggtgaa g t t c c c t g t g tgtctgtaaa atatgaacaa ggattcatga t t t g t a a a t g c t g t t t a t t t a t t g a t t g c t t c t t t c c a a a atcgaaaaaa aaaa Notch-1 C D N A from N C B I accession number z l 1886. Shaded underlined sequence is that used to design PCR primers for Notch-1. Region amplified is 622 bases. Bold italic sequence was used to design oligonucleotide probe. Jagged-1 cDNA cgggcagagg tggaagaggg gggagcgcct caaagaagcg atcagaataa taaaaggagg ccgggctctt t g c c t t c t g g aacgcgcggc tcttgaaagg gcttttgaaa agtagtgttg t t t t c c a g t c gtgcatgctc caatccacgg agtatattag agccgggacg cggcggccgc gggggcagcg acgacggcag cctcggcggg agcaccagcg ctagcagcgg cggcggcgtcl cggagtgccc gtggcgcgcg gcgcagcgat gcggtcccca cggacgcgcg gccggcccgg gcgccccctg agtcttctgc tcgccctgct ctgtgccctg cgagccaagg tgtgcggggc ctcgggtcag tttgagctgg agatcctgtc catgcagaac gtgaatggag agctacagaa tgggaactgt tgtggtggag tccggaaccc tggcgaccgc aagtgcaccc gcgacgagtg tgatacgtac ttcaaagtgt gcctcaagga gtatcagtcc cgcgtcactg ccgggggacc ctgcagcttc ggctcagggt ctacgcctgt catcgggggt aacaccttca atctcaaggc cagccgtggc aacgaccgta atcgcatcgt a c t g c c t t t c a g t t t c g c c t ggccgaggtc c t a c a c t t t g ctggtggagg cctgggattc cagtaatgac actattcaac ctgatagcat aattgaaaag g c t t c t c a c t caggcatgat aaaccctagc cggcaatggc agacactgaa acaaaacaca gggattgccc acttcgagta tcagatccga gtgacctgtg atgaccacta c t a t g g c t t t ggctgcaata agttctgtcg tcccagagat g a c t t c t t t g gacattatgc ctgtgaccag aacggcaaca aaacttgcat ggaaggctgg atgggtcctg attgcaacaa agctatctgc cgacagggct gcagtcccaa gcatgggtct tgtaaacttc caggtgactg caggtgccag tacggttggc agggcctgta ctgcgacaag tgcatcccgc acccaggatg tgtccacggc acctgcaatg aaccctggca g t g c c t c t g t gagaccaact ggggtggaca gctctgtgac aaagatctga attactgtgg gactcatcag c c c t g t c t c a accggggaac atgtagcaac actgggcctg acaaatacca gtgctcctgc ccagagggct actcgggccc caactgtgaa attgctgagc a t g c t t g t c t ctctgacccc tgccataacc gaggcagctg caaggagacc tcctcaggct ttgagtgtga g t g t t c t c c a ggctggactg gccccacgtg ttccacaaac atcgatgact gttctccaaa t a a c t g t t c c catgggggca cctgccagga tctggtgaat ggattcaagt gtgtgtgccc gccccagtgg actggcaaga c t t g t c a g t t agatgcaaat gagtgcgagg ccaaaccttg tgtaaatgcc 84 agatcctgta agaatctgat tgccagctac tactgtgatt gccttcctgg ctggatgggt cagaactgtg acataaatat caatgactgc cttggccagt gtcagaatga cgcctcctgt cgggatttgg t t a a t g g t t a t c g c t g t a t c tgtccacctg gctatgcagg cgatcactgt gagagagaca tcgatgagtg tgctagcaac cc c t g c t t g a atgggggtca ctgtcagaat gaaatcaaca gattccagtg t c t c t g t c c c a c t g g t t t c t ctggaaacct ctgtcagctg gacatcgatt actgcgagcc caacccttgc cagaatggcg cccagtgcta caatcgtgcc agtgactatt tctgcaagtg ccccgaggac tatgagggca agaactgctc acacctgaaa gaccactgcc gtaccaccac ctgcgaagtg attgacagct gcactgtggc catggcctcc aacgacacgc ctgaaggggt gcggtatatc t c t t c t a a c g tctgtggtcc ccatgggaag tgcaagagcc agtcgggagg caaattcacc tgtgactgta acaaaggctt caccggcacc tactgccatg aaaatatcaa cgactgcgag agcaacccct gtaaaaacgg tggcacctgc atcgatggcg t t a a c t c c t a caagtgtatc tgtagtgacg gctgggaggg agcgcactgt gagaacaaca taaatgactg tagccagaac c c t t g t c a c t acgggggtac atgtcgagac ctggtcaatg a c t t t t a c t g tgactgcaaa aatggctgga aaggaaagac t t g c c a t t c c cgtgacagcc agtgtgacga agccacgtgt aataatggtg gtacctgcta tgatgaagtg gacacgttta agtgcatgtg tcccggtggc tgggaaggaa caacttgtaa tatagctaga aacagtagct gcctgccgaa c c c c t g t c a t aatggaggta cctgcgtggt caatggagac t c c t t c a c c t gtgtctgcaa agaaggctgg gaggggccta t t t g t a c t c a aaataccaac gactgcagtc c c c a t c c t t g ttacaatagc gggacctgtg tggacggaga caactggtat cggtgcgaat gtgccccggg t t t t g c t g g g ccagactgca ggataaacat caatgagtgc cagtcttccc c t t g t g c c t t tggggccacc tgtgtggatg agatcaatgg ctaccagtgt a t c t g c c c t c caggacatag tggtgccaag tgccatgaag tttcagggcg a t c t t g c a t c accatgggga gagtgatact tgatggggcc aagtgggatg atgactgtaa cacctgccag tgcctgaatg gacgggtggc ctgctccaag gtctggtgtg gcccgagacc ttgcaggctc cacaaaagcc acaatgagtg ccccagtggg cagagctgca tcccggtcct ggatgaccag t g t t t c g t g c gcccctgcac tggtgttggc gaatgtcggt cctccagcct ccagccagtg aagaccaagt gcacatctga c t c c t a t t a c caggataact gtgcaaacat c a c t t t c a c c tttaacaaag agatgatgtc tccaggtctt accaccgaac acatttgcag cgaattgagg aatttgaata tcctgaagaa t g t t t c t g c t gaatattcga tctacatagc ctgtgagcct tccctgtcag caaacaatga aatacacgtg gccatctctg cagaagacat ccgggatgat gggaaccctg tcaaggaaat taccgataaa ataatagatc tcgttagtaa acgggatgga aacagctcac t t a t t g c t g c ggttgcagaa gtcagagttc agaggcgtcc tctgaaaaac agaacagatt tc c t g g t t c c tctgctgagc t c t g t c t t a a cagtggcttg ggtctgttgc ttggtgacag c c t t c t a c t g gtgtgtacgg aagcggcgga agcccagcag ccacactcac tccgcccccg aggacaacac caccaacaat gtgcgggagc agctgaacca aatcaaaaac cccatcgaga^a^cacg^gclSalicacgg^ cccattaagg attacgagaa caaaaactcc aaaatgtcaa aaatcaggac acacaactcg gaagtggagg aggatgacat ggataaacac cagcagaaag t c c g c t t t g c caaacagcca gtgtatacgc tggtagacag agaggagaag gcccccagcg gcacgccgac aaaacacccg aactggacaa ataaacagga caacagagac ttggaaagtg cccagagctt gaaccggatg gaatacatcg tatagcagac agtgggctgc cgccataggt agagtttgag ggcaccgcgg gccg Jagged-1 CDNA from NCBI accession number NM_013822. Shaded underlined sequence is that used to design PCR primers for Jagged-1. Region amplified is 900 bases. Bold italic sequence was used to design oligonucleotide probe. 

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