Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Evaluation of a fish gene transfer system : expression, fate, and germline transmission of CAT recombinant… Chong, Samuel Siong Chuan 1988

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1989_A6_7 C46.pdf [ 4.31MB ]
Metadata
JSON: 831-1.0097426.json
JSON-LD: 831-1.0097426-ld.json
RDF/XML (Pretty): 831-1.0097426-rdf.xml
RDF/JSON: 831-1.0097426-rdf.json
Turtle: 831-1.0097426-turtle.txt
N-Triples: 831-1.0097426-rdf-ntriples.txt
Original Record: 831-1.0097426-source.json
Full Text
831-1.0097426-fulltext.txt
Citation
831-1.0097426.ris

Full Text

EVALUATION OF A FISH GENE TRANSFER SYSTEM: EXPRESSION, FATE, AND GERMLINE TRANSMISSION OF CAT RECOMBINANT PLASMID AND PHAGE SEQUENCES MICROINJECTED INTO NEWLY FERTILIZED EGGS OF THE JAPANESE MEDAKA, Orvzias latipes (TEMMINCK & SCHLEGEL) By SAMUEL SIONG CHUAN CHONG B.Sc, The National University of Singapore, SINGAPORE, 1985 B.Sc, (Hons), The National University of Singapore, SINGAPORE, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1988 © Samuel Siong Chuan Chong, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Z o o l o g y  The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT The c r e a t i o n o f ' t r a n s g e n i c ' a n i m a l s has p r o v i d e d i n s i g h t s i n t o mechan i sms o f gene r e g u l a t i o n , as w e l l as opened up a new avenue f o r g e n e t i c improvement o f l i v e s t o c k , i n c l u d i n g f i s h . I n t h i s t h e s i s , t h e s u i t a b i l i t y o f t h e J a p a n e s e r i c e f i e l d f i s h o r 'medaka ' ( O r y z i a s l a t i p e s ) as a gene e x p r e s s i o n s y s t e m was e v a l u a t e d . The p r o c a r y o t i c c h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e (CAT) gene r e g u l a t e d by a doub l e e u c a r y o t i c p r o m o t e r - e n h a n c e r r e g i o n was c h o s e n as a r e p o r t e r . T h i s r e p o r t e r was i n t r o d u c e d as e i t h e r a s u p e r c o i l e d o r l i n e a r r e c o m b i n a n t p l a s m i d (pUSVCAT) , as a phage , o r as p u r i f i e d phage DNA. DNA o r phage was m i c r o i n j e c t e d i n t o t h e c y t o p l a s m o f n e w l y f e r t i l i z e d medaka eggs a t t h e 1-2 c e l l s t a g e . E x p r e s s i o n and f a t e o f t h e i n j e c t e d DNA o r phage were m o n i t o r e d b y h a r v e s t i n g medaka a t v a r i o u s d e v e l o p m e n t a l s t a g e s and p e r f o r m i n g CAT enzyme a s s a y s and S o u t h e r n b l o t a n a l y s e s , r e s p e c t i v e l y . S e v e r a l i n j e c t e d eggs were a l l o w e d t o d e v e l o p t o s e x u a l m a t u r i t y , and t h e i r o f f s p r i n g were p o o l e d and t e s t e d by CAT enzyme a s s a y f o r i n h e r i t a n c e o f t h e CAT s e q u e n c e s . The p a t t e r n s o f e x p r e s s i o n o f i n j e c t e d s u p e r c o i l e d and l i n e a r pUSVCAT DNA were v e r y s i m i l a r , i n d i c a t i n g t h a t DNA c o n f o r m a t i o n does n o t a f f e c t t he e f f i c i e n c y o f e x p r e s s i o n . CAT enzyme a c t i v i t y was d e t e c t i b l e f r om t he e a r l y h i g h b l a s t u l a s t a g e (4 h r p o s t - i n j e c t i o n ) , was s t r o n g e s t a t t h e l a t e g a s t r u l a / e a r l y n e u r u l a s t a g e (1 day p o s t - i n j e c t i o n ) , and was s u s t a i n e d b u t s l i g h t l y weake r i n t h e one-week o l d embryo . E x p r e s s i o n was s i g n i f i c a n t l y r e d u c e d i n h a t c h l i n g s (2 weeks p o s t - i n j e c t i o n ) , v a r y i n g n o t i c e a b l y among the i n d i v i d u a l s a n a l y s e d . CAT e x p r e s s i o n was s t i l l d e t e c t i b l e i n f r e e - s w i m m i n g f i s h (4 weeks p o s t - i n j e c t i o n ) . Recomb inan t CAT phage p a r t i c l e s o r p u r i f i e d CAT phage DNA were a l s o a b l e t o e x p r e s s t h e CAT gene up t o t h e f r e e - s w i m m i n g f i s h s t a g e . However , i n t h e s e t r e a t m e n t s , t h e s t r o n g e s t CAT e x p r e s s i o n was i i i seen i n the one-week old embryo instead of i n the gastrula/neurula, r a i s i n g the p o s s i b i l i t y of a role played by different vector sequences on gene expression. Studies on the fate of injected supercoiled and l i n e a r pUSVCAT revealed conversion of the input forms to high molecular weight head-to-tail and randomly oriented concatemers respectively. Total plasmid DNA increased rapidly during cleavage and gastrulation, indicative of plasmid r e p l i c a t i o n , whereas degradation of plasmid sequences was observed by the early high b l a s t u l a stage. In the gastrula/neurula derived from i n j e c t i o n of supercoiled pUSVCAT, t o t a l plasmid DNA increased ten-fold, whereas i n j e c t i o n of line a r pUSVCAT resulted i n a 12-fold increase at the same stage. In both cases, most of the observed increase was contributed by the high molecular weight concatemers. The amount of plasmid DNA decreased after the gastrula/neurula stage, and th i s DNA was exclusively of the high molecular weight form at hatching and could p e r s i s t to the free-swimming stage. Neither the DNA from injected CAT phage p a r t i c l e s nor the injected p u r i f i e d CAT phage DNA appeared to be concatenated during early embryogenesis. In both cases, however, the phage DNA appeared as higher molecular weight DNA by the one-week old embryonic stage, probably formed by covalent end-to-end l i g a t i o n s . DNA of CAT phage p a r t i c l e s did not increase u n t i l after the early high b l a s t u l a stage, but by the f l a t b l a s t u l a stage (10 hr post-injection) a three-fold increase over the input amount was observed. There was no s i g n i f i c a n t increase at the gastrula/neurula stage, nor was there an immediate decrease thereafter. Injected p u r i f i e d CAT phage DNA increased through the stages of cleavage and gastrulation, the gastrula/neurula having seven-fold more CAT phage DNA than that injected, and decreased thereafter. Both DNA of injected phage p a r t i c l e s and injected phage DNA could p e r s i s t to the free-swimming stage. i v CAT gene expression was detected i n a number of pooled offspring from several DNA and phage-treated f i s h , indicating inheritance of the input sequences. The data i n this study suggest that the germline-positive parents are probably mosaic for the presence of the CAT sequences, and that germline transmission i s possible with plasmid DNA of both conformations, DNA-carrying phage p a r t i c l e s , or p u r i f i e d phage DNA. The above r e s u l t s , coupled with the ease of handling and manipulation of the medaka embryo, strongly favour the use of the medaka as a transient expression and transgenic animal model. V TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 MATERIALS AND METHODS 6 RESULTS 1. Studies of CAT reporter gene expression during medaka development. 12 > 1.1 CAT gene expression due to cytoplasmic i n j e c t i o n of recombinant plasmid i n supercoiled and li n e a r conformation. 12 1.2 CAT gene expression due to cytoplasmic i n j e c t i o n of CAT recombinant phage p a r t i c l e s and p u r i f i e d CAT phage DNA. ">• 15 1.3 Onset of CAT gene expression i n the early medaka embryo. 18 2. Studies on the fate of introduced CAT DNA sequences during medaka development. 18 2.1 Conformational changes of injected supercoiled and lin e a r plasmid DNA. 18 2.1.1 Nature of high molecular weight pUSVCAT form. 27 2.2 Conformational changes of DNA introduced within recombinant phage p a r t i c l e s and of p u r i f i e d phage DNA. 30 2.3 Replication of injected foreign DNA sequences i n early embryos. 33 3. Inherited expression of CAT reporter gene i n o f f s p r i n g . 36 DISCUSSION 48 REFERENCES 59 LIST OF TABLES v i Table 1 Table 2 Inherited expression of supercoiled pUSVCAT DNA i n offspring. Inherited expression of l i n e a r pUSVCAT DNA i n offspring. Page 44 45 Table 3 Inherited CAT expression i n offspring of parents derived from f e r t i l i z e d eggs injected with recombinant CAT phage p a r t i c l e s . 46 Table 4 Inherited CAT expression i n offspring of parents derived from f e r t i l i z e d eggs injected with p u r i f i e d CAT phage DNA. 47 LIST OF FIGURES Diagrammatic representation of recombinant pUSVCAT DNA and recombinant CAT phage DNA. CAT gene expression due to cytoplasmic i n j e c t i o n of recombinant supercoiled and lin e a r pUSVCAT DNA. CAT gene expression due to cytoplasmic i n j e c t i o n of CAT phage p a r t i c l e s and p u r i f i e d CAT phage DNA. E a r l i e s t appearance of CAT gene expression after i n j e c t i o n of supercoiled and l i n e a r pUSVCAT DNA. Fate of injected supercoiled and li n e a r pUSVCAT DNA during medaka development. Early fate of injected supercoiled and l i n e a r pUSVCAT molecules. R e s t r i c t i o n fragment analysis of high molecular weight pUSVCAT DNA. Fate of CAT phage DNA during medaka development after i n j e c t i o n of CAT phage p a r t i c l e s and p u r i f i e d CAT phage DNA. Replication and subsequent decrease of input pUSVCAT DNA during early development. Replication and subsequent decrease of input CAT phage DNA during early development. Inherited CAT gene expression i n offspring of pUSVCAT DNA-treated parents. Inherited CAT gene expression i n offspring of parents treated with CAT phage p a r t i c l e s and p u r i f i e d CAT phage DNA. ACKNOWLEDGEMENTS v i i i I wish to express my sincere appreciation: to my supervisors, Dr. Juergen V i e l k i n d (Department of Pathology and Environmental Carcinogenesis Unit, B.C. Cancer Research Centre) and Dr. David Randall (Department of Zoology), for the i r support and guidance throughout my project; to the members of my advisory committee, Dr. Hugh Brock (Department of Zoology) and Dr. Edward Donaldson (Fisheries and Oceans, Canada), for their concern, encouragement, and help; to Dr. Bahram Sadaghiani for help with embryo microinjections; and to Ms. Barbara Schmidt, Mr. Bruce Woolcock, and Ms. Arlene Sullivan, for expert technical advice. I would s p e c i a l l y l i k e to thank the Canadian International Development Agency for providing generous f i n a n c i a l support. This research was also supported by grants from the National Institutes of Health (USA) and the Medical Research Council (Canada) to Juergen R. V i e l k i n d . INTRODUCTION 1 The Xenopus oocyte and f e r t i l i z e d egg have been successfully employed as transient expression systems i n the analysis of the temporal and s p a t i a l regulation of gene expression during early development. For example, the oocyte has been used to decipher the t r a n s c r i p t i o n a l regulation of Xenopus genes coding for 4S tRNA and 5S rRNA, and of sea urchin histone genes (reviewed i n E t k i n , 1982) ; the f e r t i l i z e d egg has been used to analyse the temporal regulation of a Xenopus gastrula-specific gene (Krieg and Melton, 1985) and the t i s s u e - s p e c i f i c regulation of a Xenopus a c t i n gene (Wilson et al., 1986). Also commonly used i n transient expression analyses i s the f e r t i l i z e d sea urchin egg, i n which ontogenic regulation of sea urchin genes coding for cytoskeletal a c t i n (Davidson et al. , 1985; Flytzanis et al. , 1987; Franks et al., 1988; Katula et al., 1987), and early and late histones (Colin et al., 1988; V i t e l l i et al. , 1988), has been demonstrated. S p a t i a l l y correct expression of a cytoskeletal a c t i n gene was also observed i n t h i s system (Hough-Evans et al., 1987, 1988). Two major characteristics of transient expression systems, mosaicism and gradual loss of the introduced gene over time, l i m i t the scope of investigations to which they can be applied. Hough-Evans et al. (1988) point out, however, that mosaicism may i n certain circumstances be advantageous since the presence of incorporated DNA only i n a percentage of c e l l s impinges less on the v i a b i l i t y of an animal because competitive depression of endogenous genes i s a l l e v i a t e d . Nevertheless, only animals which stably r e t a i n at least one copy of the gene i n every c e l l are amenable, for example, to studies of c e l l lineage or phenotypic effects of i n s e r t i o n a l mutagenesis, among others (reviewed i n Jaenisch, 1988). Integration of introduced genes and inheritance by progeny, i . e . , the creation of stable transgenic lines has been reported for several organisms, for example mice (reviewed i n Palmiter 2 and B r i n s t e r , 1986, and Jaenisch, 1988), Drosophila (Rubin and Spradling, 1982; Spradling and Rubin, 1982, 1983), and Caenorhabditis ( F i r e , 1986), and have proven invaluable i n such studies. Transgenic technology has also been applied to the genetic manipulation of commercial liv e s t o c k . Cloned growth hormone genes have been introduced into c a t t l e (McEvoy et al., 1987; King and Wall, 1988), sheep (Hammer et al., 1985; Nancarrow et al., 1987), pigs and rabbits (Hammer et al., 1985), and f i s h (Zhu et al., 1985, 1986; Chourrout et al., 1986; Dunham et al., 1987; Guyomard et al., 1988). The transfer of a disease resistence gene into poultry (Crittenden and Salter, 1985) and of a cold temperature tolerance gene into f i s h (Hew et al., 1987) have also been reported. In many instances, successful integration and or expression of the transferred genes has been demonstrated. The recent reports of gene transfer into f i s h (reviewed i n Maclean et al., 1987) have been concerned with improvement of certain t r a i t s i n commercially important species, such as enhancement of growth and tolerance of low temperatures. The aim of the present study was to evaluate the f e a s i b i l i t y of using the Japanese medaka Orvzias l a t i p e s as a transient, and also as a stable, expression system for testing developmentally important f i s h genes, or any other genes with possible applications to the genetic engineering of commercial f i s h species. The medaka appears to be an excellent model for such studies since i t s biology, including embryonic development and physiology, has been extensively studied, hundreds of eggs may be obtained dai l y (Yamamoto, 1967, 1975), and a transparent chorion permits easy observation of embryonic development. In one of the e a r l i e r gene transfer attempts into f i s h , Ozato et al. (1986) microinjected the chicken 5 - c r y s t a l l i n gene into the germinal v e s i c l e of medaka oocytes because of the d i f f i c u l t y i n locating the nucleus i n f e r t i l i z e d eggs. However, the female had to be s a c r i f i c e d to obtain the oocytes, and several other manipulations before and 3 after microinjection were required. In the present study, a s i m p l i f i e d procedure of microinjection into the cytoplasm of f e r t i l i z e d medaka eggs prior, to or immediately after f i r s t cleavage (one to two c e l l stage embryo) was adopted. The chloramphenicol acetyltransferase (CAT) gene was chosen as a reporter gene because simple and rapid tests are available for testing CAT a c t i v i t y , and because no si m i l a r enzyme has been found i n eucaryotic systems. A double v i r a l promoter-enhancer consisting of the Simian virus 40 (SV 40) early region and the long terminal repeat (LTR) of the Rous sarcoma virus (RSV) was chosen to regulate transcription of the CAT gene (Karlsson et al., 1985) because of " i t s high CAT expression i n many c e l l l i n e s as compared to other CAT constructs (Vielkind and Vogel, 1988). A number of reports have compared the expression of exogenous genes when applied i n various conformations i n vivo. Etkin and B a l c e l l s (1985) found that supercoiled DNA injected into Xenopus embryos exhibited higher expression than did l i n e a r DNA, but c o n f l i c t i n g observations were made by Wilson et al. (1986) . To test whether DNA topology has any effect on e f f i c i e n c y of gene expression i n the medaka embryo, the CAT t r a n s c r i p t i o n a l unit encompassed i n recombinant plasmid was injected i n either supercoiled or l i n e a r conformation, and CAT expression monitored during medaka development. The expression of a recombinant CAT phage, the genome of which contains three CAT transcriptional u n i t s , was also evaluated since c e l l transfection studies by Ishiura et al. (1982) and Okayama and Berg (1985) have shown that phage particle-mediated gene transfer resulted i n higher transformation rates i n mouse c e l l l i nes than did DNA-mediated gene transfer, presumably because the phage coat protects the exogenous DNA from degradation by DNases. A d d i t i o n a l l y , recombinant phage technology allows larger genes (15 to 20 kb) to be cloned, and phage are commonly used i n cloning genomic l i b r a r i e s . Successful expression of the DNA carried by the injected phage p a r t i c l e s not only would allow long stretches of genomic DNA to be tested for the effects of various introns and of distant 4 regulatory regions on gene transcription and tra n s l a t i o n (Bendig and Williams, 1983), but also would obviate the need to use p u r i f i e d phage DNA. CAT phage DNA p u r i f i e d from the recombinant CAT phage was also tested for expression i n medaka embryos since Wilson et al. (1986) reported poor tr a n s c r i p t i o n for recombinant phage DNA, possibly due to interference by the arms of the phage vector. Several other studies have concentrated on the fate of exogenous DNA injected into the f e r t i l i z e d egg, since correct temporal or s p a t i a l expression depends, i n part, on the survival and persistence of the DNA for a s u f f i c i e n t period of time for the gene to be regulated. I t has been observed that linear plasmid molecules, when injected into the cytoplasm of f e r t i l i z e d Xenopus eggs, p e r s i s t for a longer period than do supercoiled molecules (Etkin et al., 1984; Wilson et al., 1986; Marini et al., 1988). In addition, Wilson et al. (1986) observed that supercoiled DNA remained unchanged whereas li n e a r plasmid DNA was processed into high molecular weight concatenates. Marini et al. (1988), however, reported that both supercoiled and l i n e a r plasmid molecules were converted to high molecular weight species, the supercoiled DNA-derived species consisting of head-to-tail tandem arrays and the li n e a r plasmid-derived species consisting of either head-to-tail or random arrays. In sea urchins, McMahon et al. (1985) observed that only l i n e a r , but not supercoiled, DNA was processed when injected into the cytoplasm of f e r t i l i z e d eggs. Wilson et al. (1986) also reported that a A clone carrying a Xenopus cardiac actin gene persisted poorly i n the Xenopus embryo unless the insert was separated from the vector arms pr i o r to i n j e c t i o n . To determine i f DNA conformation, phage p a r t i c l e packaging or vector sequences have an effect on DNA v i a b i l i t y and processing i n the medaka embryo, supercoiled or li n e a r pUSVCAT DNA, recombinant CAT phage p a r t i c l e s , or p u r i f i e d CAT phage DNA were injected into f e r t i l i z e d medaka eggs and t h e i r fates monitored during development. 5 Aside from transient expression studies, another major objective i n gene transfer experiments i s the production of stable transgenic animals, i . e . animals i n which injected exogenous DNA i s incorporated into the host genome and i s transmitted through the germline to offspring. In mice, i t has been shown that only microinjections into the pronuclei of the f e r t i l i z e d egg resulted i n high frequencies of DNA integration, and nuclear injections resulted i n more e f f i c i e n t integration when li n e a r DNA molecules rather than supercoiled molecules were used (see Brinster et al., 1985). Also, Costantini and Lacy (1981) demonstrated that pronuclear microinjection of a A DNA clone resulted i n high frequency of integration into mouse tissues and subsequent germline transmission. In contrast to the almost absolute necessity for nuclear injections i n mice, genomic integration of a cytoplasmically injected l i n e a r plasmid DNA has been shown i n the sea urchin (Flytzanis et al., 1985), and Etkin and Pearman (1987) detected germline transmission of a supercoiled plasmid to offspring by a Xenopus adult male derived from cytoplasmic i n j e c t i o n of the exogenous DNA. Recently, Guyomard et al. (1988) and Stuart et al. (1988) were able to demonstrate that l i n e a r plasmid DNA injected into the cytoplasm of f e r t i l i z e d trout or zebrafish eggs persisted i n adult f i s h and was inherited by a certain percentage of o f f s p r i n g . V i e l k i n d et al. (1988) also reported that supercoiled pUSVCAT DNA cytoplasmically injected into f e r t i l i z e d zebrafish eggs resulted i n stable transformants whose offspring not only inherited the foreign sequences but also expressed the CAT gene. To determine i f cytoplasmic injections of supercoiled or l i n e a r pUSVCAT DNA, recombinant CAT phage, or p u r i f i e d CAT phage DNA would r e s u l t i n stable germline-positive transformants, offspring from DNA or phage-treated parents were analysed by CAT enzyme assay for inherited expression of the CAT gene. 6 MATERIALS AND METHODS Egg c o l l e c t i o n and embryo culture Clusters of f e r t i l i z e d medaka eggs attached to the females were collected 1 to 2 hr after the st a r t of the l i g h t cycle and were transferred to and maintained i n Ringer's solution (0.75% NaCl, 0.02% KC1, 0.02% CaC^, pH 7.3; Yamamoto, 1961) for up to 2 hr at 12 °C pr i o r to i n j e c t i o n to slow down the cleavage process. Injected embryos were reared i n medium consisting of 1% NaCl, 0.03% KC1, 0.04% CaCl2.2H20, 0.163% MgS0^.7H20, 0.001% methylene blue (Kirchen and West, 1976). Staging of medaka embryos was according to Matsui (1949). Embryos that hatched were transferred to and maintained i n normal tank water u n t i l analysed. Recombinant CAT gene constructs Figure 1 i l l u s t r a t e s the CAT gene constructs used i n this study. The tra n s c r i p t i o n a l unit consists of the b a c t e r i a l CAT coding sequence under the regulation of the Rous sarcoma virus (RSV) LTR and Simian virus 40 (SV 40) early region double promoter-enhancer regions. This CAT tra n s c r i p t i o n unit was tested as a recombinant plasmid pUSVCAT (Karlsson et al., 1985) i n supercoiled form and also as a li n e a r molecule after digestion with S a i l . A recombinant CAT phage p a r t i c l e containing three tandem copies of the CAT tran s c r i p t i o n unit was also used i n th i s study (Vielkind and Vogel, 1988), as was the p u r i f i e d DNA from this recombinant phage. pUSVCAT DNA was extracted by using a modified B r i j detergent method (Clewell and H e l i n s k i , 1969) and was p u r i f i e d by two successive CsCl-ethidium bromide equilibrium density centrifugations. Recombinant CAT phage were p u r i f i e d from plate lysates by CsCl step gradient centrifugation and were dialysed against SM buffer (100 mM NaCl, 8 mM MgS04.7H20, 50 mM Tris pH 7.5, 7 Figure 1 (a) Plasmid map of pUSVCAT showing CAT coding region, promoter-enhancer regions (*•-), and relevant r e s t r i c t i o n s i t e s . (b) pUSVCAT lin e a r i z e d at the S a i l s i t e . (c) Map of recombinant CAT phage DNA. The insert consists of three tandem copies of a 5200 bp BamHI fragment of pUSVCAT containing the CAT transcription u n i t . An, polyadenylation s i t e ; ampr, a m p i c i l l i n resistance; K, Kpnl r e s t r i c t i o n s i t e ; S, S a i l r e s t r i c t i o n s i t e ; B, BamHI r e s t r i c t i o n s i t e . SV40or i pUC9 An RSV SV 40 cat LTR ori K amp'' B EMBL3 arm 3 x BamHI fragments I I EM3L3 arm B B B B CAT phage DNA (44.2 kb) 9 0.01% g e l a t i n ) . CAT phage DNA was extracted from some of the phage as described i n Maniatis et al. (1982). Microinjection Supercoiled or line a r pUSVCAT DNA, recombinant CAT phage p a r t i c l e s , or p u r i f i e d CAT phage DNA were microinjected into the cytoplasm of the medaka zygote p r i o r t o , or immediately a f t e r , f i r s t cleavage (1-2 c e l l stage embryo), by using a b o r o s i l i c a t e glass c a p i l l a r y needle (3 to 5 /im diameter) mounted on a micromanipulator. Injections were done under a binocular microscope (Zeiss) with a magnification range of 8x to 50x. Concentrations of 50 //g/ml plasmid DNA, 8 x 1 0 ^ phage particles/ml (equivalent to 5 /ig/ml phage DNA) , and 5 jug/ml or 20 Mg/n»l p u r i f i e d phage DNA were used. Phenol red had been added to the DNA/phage solutions to a f i n a l concentration of 0.25% to aid i n estimation of i n j e c t i o n volume (ca. 500 p i ) . CAT assay CAT assays were performed e s s e n t i a l l y as described by Gorman et al. (1982). Individual embryos, hatchlings, and free-swimming f i s h or pools of three or f i v e embryos or hatchlings were homogenized i n 100 nl 250 mM Tris pH 8.0, and then subjected to three 5 min cycles of freeze-thawing; extracts were obtained after centrifugation (Eppendorf, 5 min, 4 °C) . To 100 extract, 20 Hi d H2° . 2 A*l 1 4C-chloramphenicol (NEN DuPont, 60 mCi/mmol, 100 /id/ml), and1 20 / i l 4 mM acetyl coenzyme A (Boehringer Mannheim) were added, and the mixture was incubated for 1 hr at 37 °C. The ^C-chloramphenicol and i t s acetylated forms were extracted with 1 ml ethyl acetate, dried under vacuum, resuspended i n 30 / i l ethyl acetate, and then spotted and separated on s i l i c a gel chromatography plates (J.T. Baker Co.) for 50 min i n chloroform:methanol (95:5). After the plates were a i r - d r i e d , autoradiograms were produced by 10 exposure of X-ray f i l m (Kodak XAR-5) to these plates for one or seven days i n the presence of an intensifying screen (DuPont). DNA extraction Individual embryos, hatchlings, and free-swimming f i s h , as well as pools of ten embryos, were homogenized i n 200 pi of l x SET (100 mM NaCl, 20 mM EDTA, 50 mM Tris pH 7.8), 0.5% SDS, 0.5 mg/ml proteinase K and incubated for 2 to 4 hr at 37 °C. The samples were then extracted with one volume of phenol:chloroform:isoamyl alcohol (25:24:1), re-extracted with an equal volume of butanol:isopropanol (7:3), precipitated with 2 volumes of 95% ethanol for at least 2 hr at -20 °C and redissolved overnight i n TE (10 mM Tris pH 8.0, 1 mM EDTA). Determination of embryo DNA content The genomic DNA content of medaka embryos at stages up to the early high b l a s t u l a stage was calculated by multiplying the amount of DNA present per d i p l o i d medaka c e l l (see Uwa and Iwata, 1981) to the estimated number of c e l l s present at the embryonic stage. For l a t e r developmental stages, DNA was extracted from embryos and measured fluorometrically using the bis-benzimidazole (Hoechst 33258, Hoefer S c i e n t i f i c Instruments) method as specified by the manufacturer. Southern blots Total DNA from individual embryos, hatchlings, or free-swimming f i s h , or aliquots of DNA from pooled samples (equivalent to single embryos of the various stages), either non-digested or completely digested by r e s t r i c t i o n enzymes as specified by the manufacturer, were subjected to electrophoresis i n 0.8% agarose (Bio-Rad) gels. Gels were soaked once i n 250 mM HC1 for 10 min to p a r t i a l l y hydrolyse DNA, twice i n 1.5 M NaCl, 0.5 M NaOH for 15 min to 11 denature DNA strands, and twice i n 1.5 M NaCl, 0.5 M Tris pH 7.5 for 15 min to neutralize the pH of the g e l . After c a p i l l a r y transfer of DNA onto nylon f i l t e r s (Schleicher & Schuell Nytran) i n 20x SSC buffer (3 M NaCl, 0.3 M sodium c i t r a t e , pH 7.0), the f i l t e r s were dried in vacuo for 2 hr at 80 °C. Hybridizations F i l t e r s were prehybridized for 15 min at 60 °C with a solution containing 3x SSC, 10 mM Tris pH 7.6, 10 mM EDTA, 0.5% SDS, l x Denhardt's (0.02% BSA, 0.02% polyvinylpyrollidone, 0.02% F i c o l l ) , and 0.1 mg/ml yeast RNA. The f i l t e r s were then hybridized overnight at 60 °C i n a sim i l a r solution containing pUSVCAT DNA, which had been l a b e l l e d by random hexamer priming (Feinberg and Vogelstein, 1983/84) with J/:P-dCTP to a s p e c i f i c a c t i v i t y of >5 x 10 cpm//ig. The f i l t e r s were washed twice i n 2x SSC, 0.5% SDS. for 30 min at room temperature (low stringency), and twice i n O.lx SSC, 0.5% SDS for 30 min at 60°C (high stringency). Autoradiograms of the f i l t e r s were then obtained by exposure of the dried f i l t e r s to X-ray f i l m i n the presence of inte n s i f y i n g screens. Quantitation of DNA hybridization signals Hybridization bands of each f i l t e r lane were excised and placed i n 7 ml p l a s t i c s c i n t i l l a t i o n v i a l s ; the remainder of each lane were placed i n separate v i a l s to be counted. 5 ml of a toluene (BDH chemicals) s c i n t i l l a t i o n c o c k t a i l containing 0.4% w/v PPO (2,5-diphenyloxazole; BDH chemicals) and 0.01% w/v dimethyl POPOP (1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene; Packard) was added to each v i a l , and the v i a l s counted i n a Packard s c i n t i l l a t i o n counter for 10 min each. Hybridization bands containing known amounts of DNA were also counted i n the same manner and used as a reference to convert cpm (counts per minute) values to picogram DNA amounts. 1 2 RESULTS 1. Studies of CAT reporter gene expression during medaka development CAT expression gene during medaka development was monitored by performing CAT enzyme assays on individuals or pooled batches at various stages from the early cleavages of the egg up to the free-swimming f i s h stage. 1.1 CAT gene expression due to cytoplasmic i n j e c t i o n of recombinant plasmid i n supercoiled and l i n e a r conformation. The results of the CAT enzyme assay of individual medaka of various stages derived from f e r t i l i z e d eggs injected with supercoiled pUSVCAT DNA i s shown i n Figure 2a. Altogether, ten individuals at each stage of development were assayed, of which representative results are shown. CAT enzyme a c t i v i t y was not detectible i n the 32-64 c e l l stage embryo (lane 1, 2 hr post-i n j e c t i o n ) . However, CAT enzyme a c t i v i t y i n gastrula/neurula stage embryos was very prominent (lane 2-4, 1 day p o s t - i n j e c t i o n ) , and was sustained but s l i g h t l y weaker i n the one-week old embryo (lane 5-7), a stage when the eye and most major organs are formed. Reduced CAT a c t i v i t y was observed at the time of hatching and varied noticeably among the hatchlings assayed (lane 8-10, 2 weeks po s t - i n j e c t i o n ) . By the free-swimming f i s h stage, CAT a c t i v i t y was s t i l l detectible i n a few of the f i s h assayed, one of which had a moderately strong signal (lane 11-13, 4 weeks po s t - i n j e c t i o n ) . No CAT a c t i v i t y was detected i n untreated medaka at s i m i l a r developmental stages (data not shown), arguing that the CAT enzyme a c t i v i t y i n treated embryos i s not due to expression of endogenous genes. Sleigh (1986) and Crabb and Dixon (1987) have reported the presence of substances i n c e l l extracts that interfere with CAT enzyme a c t i v i t y . As c o n t r o l , CAT assays were performed on homogenates of uninjected embryos at di f f e r e n t stages to which equal amounts of commercially available CAT enzyme had. been added. No 13 Figure 2 CAT gene expression after i n j e c t i o n of (a) supercoiled, and (b) l i n e a r , pUSVCAT DNA into f e r t i l i z e d medaka eggs. Eggs were injected with 25 pg of either plasmid form and allowed to develop u n t i l harvested. Individuals at various developmental stages were harvested and assayed for CAT enzyme a c t i v i t y , of which representative autoradiograms are shown. CM, C-chloramphenicol; Ac-j^ - and Ac-j-CM, monoacetylated forms of CM; Ac-^  3-CM, diacetylated CM. S t a g e s : 32-64 late g a s t r u l a / o n e - w e e k old hatch l ing f ree-swimming cel ls early neurula embryo f ish 15 detectible differences i n CAT a c t i v i t y signal were observed among these control groups (data not shown), indicating that the observed pattern of CAT signals obtained by i n j e c t i n g supercoiled pUSVCAT DNA i s not a result of diffe r e n t i n h i b i t o r y capacities of the embryonic stages, but represents the CAT expression pattern of the introduced gene. For embryos injected with S a i l - l i n e a r i z e d pUSVCAT (Fig 2b), an expression pattern si m i l a r to that for supercoiled pUSVCAT injected embryos was observed, but with marginally stronger signals at the gastrula/neurula stage and s l i g h t l y weaker signals at the hatchling stage. Very weak CAT expression was detected i n a few free-swimming stage f i s h (lane 11-13). 1.2 CAT gene expression due to cytoplasmic i n j e c t i o n of CAT recombinant phage p a r t i c l e s and p u r i f i e d CAT phage DNA. The results of the CAT expression experiments using CAT phage pa r t i c l e s i s shown i n Figure 3a. No CAT expression was detectible i n embryos assayed at the 32-64 c e l l stage (lane 1), but weak CAT expression was observed i n several late gastrula/early neurula stage embryos (lane 2-4). A s l i g h t l y stronger CAT expression was consistently observed i n one-week old embryos (lane 5-7), and CAT expression was either weak or not detected at the hatchling stage (lane 8-10). However, weak CAT expression was s t i l l detectible i n a few free-swimming stage f i s h (lane 11-13). Injections with p u r i f i e d CAT phage DNA resulted i n a pattern of CAT gene expression s i m i l a r to that obtained with CAT phage p a r t i c l e s (Fig 3b). However, generally stronger signals were observed, presumably res u l t i n g from the higher CAT phage DNA concentration used, which was the equivalent of five times the DNA administered through phage p a r t i c l e i n j e c t i o n s . For example, a moderately strong CAT a c t i v i t y was consistently detected i n the late gastrula/early neurula (lane 2-4), whilst one-week old embryos displayed a s i g n i f i c a n t l y stronger CAT a c t i v i t y (lane 5-7). CAT signals were generally 16 Figure 3 CAT gene expression after i n j e c t i o n of (a) CAT phage p a r t i c l e s , and (b) CAT phage DNA, into f e r t i l i z e d medaka eggs. Eggs were injected with 4 x 10^ recombinant phage p a r t i c l e s (equivalent to 2 pg CAT phage DNA) or 10 pg p u r i f i e d phage DNA and allowed to develop u n t i l harvested. Individuals at various developmental stages were harvested and assayed for CAT enzyme a c t i v i t y , of which representative autoradiograms are shown. a 1 2 3 4 5 6 7 8 9 10 11 12 13 A c 3 - C M A c r C M © © © © © © © • • t f f C M © • • • • b 1 2 3 4 5 6 7 8 9 10 11 12 13 • t f t • • • • • • • • • 6 • • • © © © © © © » • I I + + S t a g e s : 32-64 late gastrula/ o n e - w e e k old hatchl ing f ree-swimming ce l l s early neurula embryo f ish 18 weaker and varied widely i n individual hatchlings (lane 8-10), and were detectible only i n a few of the free-swimming f i s h tested (lane 11-13). 1.3 Onset of CAT gene expression i n the early medaka embryo The very strong CAT expression observed i n gastrula/neurula stage embryos derived from treatment with either supercoiled or l i n e a r pUSVCAT DNA suggest that CAT expression began at an e a r l i e r developmental stage. CAT gene expression was therefore examined between the 32-64 c e l l stage and the late gastrula/early neurula stage on pooled batches of 3 embryos (Fig 4). Expression appeared to begin from the early high b l a s t u l a stage (lanes 2, 4 hr pos t - i n j e c t i o n ) , a clear signal being apparent by the f l a t b l a s t u l a stage (lanes 3, 10 hr pos t - i n j e c t i o n ) , and a very strong CAT a c t i v i t y was observed i n the gastrula/neurula (lanes 4, 1 day pos t - i n j e c t i o n ) . No differences were observed between the supercoiled and li n e a r DNA-treated groups. 2. Studies on the fate of introduced CAT DNA sequences during medaka  development The fate of the introduced CAT DNA sequences during medaka development was monitored by performing Southern bl o t analysis of DNA samples obtained from individuals or pools at various stages from the 1-2 c e l l embryonic stage to the free-swimming f i s h stage. 2.1 Conformational changes of injected supercoiled and l i n e a r plasmid DNA The results of the Southern bl o t analyses of individual medaka DNA, ar i s i n g from experiments using supercoiled pUSVCAT DNA, i s shown i n Figure 5a. At the 32-64 c e l l stage (lane 1), a l l three forms' which are present i n pUSVCAT plasmid DNA preparations (mostly supercoils, and some open c i r c l e s and multimeric c i r c l e s ; not shown) were detected. By the late gastrula/early neurula stage an add i t i o n a l , high molecular weight form of greater than 23.1 19 Figure 4 E a r l i e s t appearance of CAT gene expression after i n j e c t i o n of (a) supercoiled, and (b) l i n e a r , pUSVCAT DNA. 25 pg of either plasmid form was injected into each f e r t i l i z e d egg. At various early embryonic stages, 3 samples were pooled together and assayed for the presence of CAT enzyme a c t i v i t y . * • • • a 20 g a s t r u l a / n e u r u l a f l a t b l a s t u l a early high blastula 3 2 - 6 4 cel ls gastrula/neurula f lat b l a s t u l a early high blastula 3 2 - 6 4 cel ls m CD o 21 Figure 5 Southern bl o t analysis of the fate of injected (a) supercoiled, and (b) l i n e a r , pUSVCAT molecules during medaka development. F e r t i l i z e d eggs were injected with 25 pg of either plasmid form. DNA was extracted from individuals at various developmental stages, subjected to electrophoresis i n 0.8% agarose gels, and transferred to nylon membranes. The blots were probed then with pUSVCAT DNA lab e l l e d by random hexamer priming, representative autoradiograms of which are shown. (c) l i n e a r pUSVCAT DNA standard, equivalent to input amount (25 pg). sc, supercoiled pUSVCAT DNA; oc, open c i r c u l a r pUSVCAT DNA; mc, multimeric c i r c u l a r pUSVCAT DNA; In, l i n e a r pUSVCAT DNA; hmw, high molecular weight pUSVCAT DNA. Lambda H i n d l l l size standards (kb) are shown at r i g h t of panel (a). 22 a 1 2 3 4 5 6 7 8 9 10 11 12 13 b 1 2 3 4 5 6 7 8 9 10 11 12 13 C In I 1 1 I 1 1 Stages: 32- 'ate one-week hatchling free-64 gastruia/ old embryo swimming cells early fish neurula 23 kb was c l e a r l y evident (lane 2-4), co-migrating with the high molecular weight f r a c t i o n of medaka DNA (as seen by ethidium bromide staining of the gel prior to Southern b l o t t i n g ; data not shown). An increase i n a l l the plasmid forms was seen, with the high molecular weight form showing the greatest increase. Strong hybridization smears were also observed, suggesting degradation of a s i g n i f i c a n t percentage of plasmid DNA. In the one-week old embryo, the open c i r c u l a r and multimeric forms could not be detected, some supercoiled plasmid was s t i l l evident, and the rest of the plasmid DNA was of the high molecular weight form (lane 5-7); plasmid DNA degradation was s t i l l evident. In hatchlings, plasmid DNA was further reduced and only the high molecular weight form remained (lane 8-10). Input plasmid DNA persisted i n one of several free-swimming f i s h analysed (lane 11-13). When l i n e a r pUSVCAT DNA was used, DNA extracted from 32-64 c e l l stage embryos contained a strongly hybridizing high molecular weight form (Fig 5b, lane 1), suggesting a f a i r l y rapid conversion of the injected l i n e a r molecules. A f a i n t smear suggests that plasmid DNA degradation had already begun. In the late gastrula/early neurula, a very strong hybridization i n the high molecular weight band was observed, indicating a large increase i n plasmid DNA. A strong hybridization smear was also apparent, suggesting substantial degradation of the plasmid DNA. Only the high molecular weight form was present i n the one-week old embryo, and plasmid degradation was s t i l l evident (lane 5-7). In hatchlings, the amount of plasmid DNA present was further reduced, and i t s continued degradation was s t i l l detected i n some samples (lane 8-10). Plasmid DNA was s t i l l c l e a r l y apparent i n one of several free-swimming stage f i s h (lane 11-13). The very strong hybridization signals already seen at the gastrula/neurula stage i n both the supercoiled and li n e a r pUSVCAT experiments suggest that processing of the injected plasmids must have begun e a r l i e r . In order to have an idea of the ra p i d i t y of plasmid conversion, embryos at 24 e a r l i e r stages between the 1-2 c e l l stage to the 30 somite neurula stage were analysed. Ten embryos at each stage were pooled, and Southern blot analysis was performed on embryo-equivalent DNA aliquots after complete digestion with Xhol, which does not recognize any s i t e on pUSVCAT DNA. Digestion with Xhol reduces high molecular weight medaka DNA to smaller fragments, thus minimizing any possible impedance of pUSVCAT DNA migration during electrophoresis. DNA extracted from 1-2 c e l l stage embryos within 5 min of i n j e c t i o n with supercoiled pUSVCAT DNA contained the expected supercoiled, open c i r c u l a r , and multimeric c i r c u l a r forms (Fig 6a, lane 1). The high molecular weight form, which was not different from that observed before, became apparent at the early high b l a s t u l a stage, and an ove r a l l increase i n the other pUSVCAT forms was seen (lane 2, 4 hr post - i n j e c t i o n ) . By the f l a t b l a s t u l a stage (lane 3, 10 hr po s t - i n j e c t i o n ) , the high molecular weight form became more prominent than the other forms, suggesting preferred r e p l i c a t i o n of the high molecular weight form. In addition, a strong smear was observed, s i g n a l l i n g the onset of plasmid DNA degradation. The strongest ov e r a l l DNA hybridization signal was observed i n the late gastrula/early neurula, as previously noted (lane 4 , 1 day post-injection) but by the 30 somite stage t o t a l plasmid DNA had s i g n i f i c a n t l y declined (lane 5, 3 days po s t - i n j e c t i o n ) . In contrast to the experiment with supercoiled pUSVCAT DNA, much of the li n e a r pUSVCAT DNA injected into f e r t i l i z e d eggs was almost immediately converted to high molecular weight form (Fig 6b, lane 1, 5 min post-i n j e c t i o n ) . In addition, f a i n t bands corresponding to supercoiled and open c i r c u l a r pUSVCAT forms could be made out, indicating some conversion of the li n e a r DNA to these forms too. In the early high b l a s t u l a stage, the amounts of supercoiled, open c i r c u l a r , and especially high molecular weight pUSVCAT forms increased while that of the li n e a r form diminished (lane 2). Multimeric pUSVCAT c i r c l e s were c l e a r l y apparent i n f l a t b l a s t u l a stage embryos (lane 3), while t o t a l plasmid DNA was further increased. The strongest DNA 25 Figure 6 Southern blot analysis of the early fate of injected (a) supercoiled, and (b) l i n e a r , pUSVCAT molecules. F e r t i l i z e d eggs injected with either supercoiled or li n e a r pUSVCAT DNA were harvested at various early embryonic stages, and t o t a l DNA was obtained from pools of ten embryos. Embryo-equivalent aliquots were subjected to electrophoresis, b l o t t e d , hybridized to radio-l a b e l l e d pUSVCAT probe, and autoradiographed as described i n Fig 5. std, standards of the various c i r c u l a r and l i n e a r pUSVCAT forms. CN CO I 8 I I O (fl in 1 CO 1 CM t i -* I CO CM 03 30 somites gastrula/neurula flat blastula early high blastula 1-2 cells 30 somites gastrula/neurula flat blastula early high blastula 1-2 cells W 0) CO CO OT 27 hybridization signal and smearing was seen i n the late gastrula/early neurula (lane 4). By the 30 somite stage (lane 5), t o t a l plasmid DNA markedly decreased and only the high molecular weight form remained. 2.1.1 Nature of high molecular weight pUSVCAT form The fact that the migration of the high molecular weight plasmid form was unaffected by digestion of the embryo DNA samples with Xhol, which reduces genomic DNA to smaller fragment sizes but does not cut pUSVCAT DNA, refutes the p o s s i b i l i t y that i t arose from entrapment of supercoiled, open c i r c u l a r , or l i n e a r plasmids i n the high molecular genomic DNA f r a c t i o n . To determine the nature of th i s high molecular weight pUSVCAT form, Southern bl o t analysis was performed on DNA from hatchlings, a stage i n which the remaining pUSVCAT DNA had e a r l i e r been observed to be exclusively of the high molecular weight form. DNA from individual hatchlings was digested with Kpnl and analysed by Southern bl o t hybridization using radioactively l a b e l l e d pUSVCAT DNA as a probe. There i s only one Kpnl r e s t r i c t i o n s i t e on pUSVCAT, and on a S a l l -l i n e a r i z e d molecule, Kpnl produces two fragments of 4.9 kb and 3.0 kb. In hatchlings derived from eggs injected with supercoiled pUSVCAT DNA, digestion of the DNA produced a single hybridization band corresponding to a 7.9 kb li n e a r monomer (Fig. 7a). This resul t suggests that the high molecular weight plasmid form derived from supercoiled plasmid injections probably consisted of tandem arrays of plasmid monomers oriented head-to-tail. However i n hatchlings derived from eggs injected with l i n e a r pUSVCAT DNA, three strong hybridization bands of approximately 9.8 kb, 7.9 kb, and 6.0 kb were seen (F i g . 7b,c). In addition, two other very weak hybridization bands of approximately 4.9 kb and 3.0 kb could be detected. The r e s t r i c t i o n pattern obtained indicates that the resident high molecular weight plasmid form consisted of randomly oriented tandem arrays of the l i n e a r plasmids. Kpnl digestion of such a tandem array would be expected to y i e l d many copies of 28 Figure 7 Southern blot analysis of high molecular weight pUSVCAT DNA pe r s i s t i n g i n hatchlings. Hatchlings derived from eggs injected with (a ) supercoiled, or (b) l i n e a r , pUSVCAT DNA were in d i v i d u a l l y harvested for t h e i r DNA. The DNA samples were digested with Kpnl, subjected to electrophoresis, b l o t t e d , hybridized to radio-l a b e l l e d pUSVCAT probe, and autoradiographed as described i n Fig 5. (c) shorter duration autoradiogram of lanes 1-3 i n panel (b). Sizes of the r e s t r i c t i o n bands are given to the l e f t of panels (a) and (b). s t d , DNA standards of 7.9kb l i n e a r pUSVCAT DNA molecule and the two fragments res u l t i n g from Kpnl digestion of S a i l l i n e a r i z e d pUSVCAT. (d) Diagram of a hypothetical multimer of S a i l l i n e a r i z e d pUSVCAT DNA showing a l l possible (head-to-tail, head-to-head, and t a i l - t o - t a i l ) l i g a t i o n products. Unlabelled v e r t i c a l l i n e s represent S a i l l i g a t i o n junctions. The predicted size (kb) of r e s t r i c t i o n fragments r e s u l t i n g from Kpnl (K) digestion are indicated. 29 T3 tn CO CN O) 0) o ^ CO I I ai OQ if) CO CM CO 05 O 05 o 0>N" CO «tf pj 9 * in CO CM O) 3 0 middle fragments with sizes represented by the three strong hybridization bands, and fewer copies of end fragments corresponding to the weak hybridization bands (Fig 7d). The r e l a t i v e l y stronger hybridizing signal of the unit length band compared to the other two middle fragment bands implies that a majority of the l i g a t i o n junctions were of the head-to-tail type. 2.2 Conformational changes of DNA introduced within recombinant phage p a r t i c l e s and of p u r i f i e d phage DNA The results of the Southern b l o t analyses of ind i v i d u a l medaka DNA, a r i s i n g from experiments using CAT phage p a r t i c l e s , i s shown i n Figure 8a. At the 32-64 c e l l stage, a band corresponding to CAT phage DNA was observed (lane 1). This band was also present i n the late gastrula/early neurula (lane 2-4) but d i f f e r e d from the preceding stage i n having a s l i g h t l y stronger hybridization signal with some very l i g h t smearing, indica t i v e of concurrent foreign DNA r e p l i c a t i o n and degradation. This observation therefore suggests that DNA contained within the injected phage p a r t i c l e s was released p r i o r to t h i s stage. In the one-week old embryo, the CAT phage DNA appeared to co-migrate with the high molecular weight f r a c t i o n of medaka DNA (lane 5-7). Overall hybridization signal was only s l i g h t l y weaker, but smears were more v i s i b l e . CAT phage DNA was further reduced i n hatchlings (lane 8-10), yet appeared to p e r s i s t through to the free-swimming f i s h stage (lane 11-13). Eggs injected with p u r i f i e d CAT phage DNA and analysed at the 32-64 c e l l stage also showed a band corresponding to unit length CAT phage DNA (Fig 8b, lane 1). By the gastrula/neurula stage, a stronger hybridization band was observed, indic a t i v e of CAT phage DNA r e p l i c a t i o n . A prominent smear was also evident, suggesting that CAT phage DNA degradation had also occurred. In the one-week old embryo, there seems to have been a s h i f t toward higher molecular weight i n the DNA hybridization band, but CAT phage DNA was reduced and degradation products were s t i l l c l e a r l y evident (lane 5-7). CAT phage DNA was 31 Figure 8 Southern bl o t analysis of the fate of (a) DNA of injected CAT phage p a r t i c l e s and (b) injected CAT phage DNA molecules during medaka development. F e r t i l i z e d eggs were injected with 4 x 10 recombinant phage p a r t i c l e s (equivalent to 2 pg CAT phage DNA) or 10 pg p u r i f i e d phage DNA. DNA was extracted from individuals at various developmental stages, subjected to electrophoresis, b l o t t e d , hybridized to radio-labelled pUSVCAT probe, and autoradiographed as described i n Fig 5. Arrows point to CAT phage DNA monomer. Lambda H i n d l l l size standards (kb) are shown at right of panel (a). 32 a 1 2 3 4 5 6 7 8 9 10 11 12 13 ^ 23.1 -« 9.4 -< 6.6 4.4 -« 2.3 2.0 b 1 2 3 4 5 6 7 8 9 10 11 12 13 4* • I 1 1 1 1 1 Stages: 3 2 ~ late one-week hatchling f ree-64 gastrula/ old embryo swimming cells early fish neurula 3 3 s i g n i f i c a n t l y reduced and no longer detectible i n several individuals at the hatchling stage (lane 8-10), but continued to per s i s t to the free-swimming stage i n at least one of the f i s h tested (lane 11-13). 2.3 Replication of injected foreign DNA sequences i n early embryos As already observed i n the Southern analyses of DNA from embryos injected with supercoiled and li n e a r pUSVCAT DNA, CAT phage DNA and CAT phage p a r t i c l e s , the t o t a l amount of plasmid DNA was greatest at the gastrula/neurula stage, decreasing s i g n i f i c a n t l y thereafter. In the experiments using supercoiled or li n e a r plasmid DNA, the increasing i n t e n s i t i e s of DNA hybridization signals from the 1-2 c e l l stage to the gastrula/neurula stage suggest that the injected sequences were replicated during the cleavage and gastrulation stages of medaka embryogenesis. At the same time, the hybridization intensity of the smears indicate that a fr a c t i o n of the hybridization signal consisted of degraded foreign DNA. In an attempt to determine the degree of foreign DNA r e p l i c a t i o n during early embryogenesis, a Southern blot hybridization assay of S a i l digested embryo-equivalent DNA was performed. This method was chosen over the simpler 'dot b l o t ' assay i n order to separate undegraded DNA from degraded DNA. For pUSVCAT injected eggs a S a i l band migrating as a 7.9 kb molecule corresponds to i n t a c t ( i . e . t r a n s c r i p t i o n a l l y functional) pUSVCAT monomers, whereas for eggs injected with CAT phage DNA, a S a i l band migrating as a 15.6 kb molecule corresponds to intact CAT insert ( i . e . 3 tandemly arranged CAT transcription u n i t s ) . In experiments using supercoiled pUSVCAT DNA, t o t a l pUSVCAT DNA increased approximately ten-fold by the late gastrula/early neurula stage (22 hr p o s t - i n j e c t i o n ) , but the amount of intact pUSVCAT molecules at this stage represented only a s i x - f o l d increase (Fig 9b & d). Of the t o t a l pUSVCAT sequences present at t h i s stage, 50% were recovered as unit length molecules. 34 Figure 9 Rapid increase and subsequent decrease of input pUSVCAT DNA during early embryogenesis, as measured by Southern bl o t hybridization assay. Embryo-equivalent aliquots of the DNA samples generated i n the experiments i n Fig 6 were digested with S a i l , subjected to electrophoresis, b l o t t e d , hybridized to radio-labelled pUSVCAT probe, and autoradiographed as described i n Fig 5 . Hybridization bands on each f i l t e r lane corresponding to unit intact l i n e a r pUSVCAT molecules were cut out and quantified by s c i n t i l l a t i o n counting. The remainder of each lane corresponding to plasmid smears were counted separately; t o t a l pUSVCAT DNA present i n each embryo was calculated from the counts obtained from the entire lane. (a) and (b) Total pUSVCAT DNA per embryo after i n j e c t i o n of f e r t i l i z e d eggs with lin e a r and supercoiled pUSVCAT DNA, respectively. (c) and (d) intact pUSVCAT DNA per embryo after i n j e c t i o n with l i n e a r and supercoiled pUSVCAT DNA, respectively. The time of harvest, embryonic stage, and genomic DNA content of each stage are indicated on the abscissa. 36 The greatest degree of increase was recorded with l i n e a r pUSVCAT DNA; t o t a l pUSVCAT DNA increased 12-fold by the gastrula/neurula stage, while the intact molecules were approximately nine-fold greater than that at the time of in j e c t i o n (Fig 9a & c ) . Intact pUSVCAT units also constituted 50% of the t o t a l pUSVCAT DNA sequences present at the gastrula/neurula stage. When CAT phage p a r t i c l e s were used, t o t a l CAT DNA increased three-fold, but intact CAT insert increased less than one-fold, by the f l a t b l a s t u l a stage (Fig 10b & d). With p u r i f i e d CAT phage DNA, t o t a l CAT DNA sequences increased approximately seven-fold by the gastrula/neurula stage; intact CAT insert increased only three-fold and accounted for roughly 30% of the t o t a l CAT DNA sequences at the gastrula/neurula stage (Fig 10a & c ) . By the 30 somite stage of embryonic development (72 hr post-injection), a l l three DNA-injected groups (supercoiled pUSVCAT, l i n e a r pUSVCAT, and CAT phage DNA), but not the CAT phage p a r t i c l e - i n j e c t e d group, had s i g n i f i c a n t l y reduced t o t a l and intact CAT DNA. These results r e f l e c t a change i n r e p l i c a t i o n of the foreign sequences r e l a t i v e to th e i r degradation, suggesting a slowdown or complete h a l t i n CAT DNA r e p l i c a t i o n after the gastrula/neurula stage, while the CAT DNA that were s t i l l present continued to be degraded. Throughout the period of embryonic development analysed (1-2 c e l l stage to 30 somite stage), the genomic DNA content of embryos continues to increase, although at diminishing rates of increase with progressing development. 3. Inherited expression of CAT reporter gene i n offspring One of the objectives i n this study was to determine i f the injected DNA or phage would r e s u l t i n genomic integration of the foreign DNA as well as i n the inheritance and expression of these sequences by progeny f i s h . In order to i d e n t i f y positive founder f i s h , several treated f i s h were out-crossed with untreated medaka. One-week old F-^  embryos or F-^  hatchlings obtained from each out-cross were harvested and pooled i n batches of f i v e and analysed for 3 7 Figure 10 Increase and subsequent decrease of input CAT phage DNA during early embryogenesis. F e r t i l i z e d eggs were injected with 4 x 10^ recombinant phage p a r t i c l e s (equivalent to 2 pg CAT phage DNA) or 2 pg p u r i f i e d CAT phage DNA. Total DNA was extracted from pools of ten embryos at various early embryonic stages. Embryo-equivalent aliquots were digested with S a i l , subjected to electrophoresis, blotted, hybridized to radio-labelled pUSVCAT probe and autoradiographed as described i n Fig 5. Hybridization bands i n each f i l t e r lane corresponding to intact CAT DNA inserts, and the remainder of each lane (smears), were separately quantified by s c i n t i l l a t i o n counting as described i n Fig 9. Total CAT DNA present i n each embryo was calculated on a sim i l a r basis as i n Fig 9. (a) and (b) Total CAT DNA per embryo after i n j e c t i o n with p u r i f i e d CAT phage DNA and recombinant CAT phage p a r t i c l e s , respectively. (c) and (d) intact CAT DNA inserts per embryo after i n j e c t i o n with CAT phage DNA and CAT phage p a r t i c l e s , respectively. 3 8 39 inherited CAT gene expression. Representative CAT assays of pooled F-^  batches from several parents derived from eggs cytoplasmically injected with supercoiled and li n e a r pUSVCAT, recombinant CAT phage, and p u r i f i e d CAT phage DNA are shown i n Figures 11 and 12. Altogether, three to four batches of F-^ , representing 15 and 20 offspring respectively, were obtained from each out-cross, and analysed for CAT expression, the results of which are l i s t e d i n Tables 1 through 4. CAT-positive offspring from parents representing a l l four i n j e c t i o n groups (supercoiled pUSVCAT, li n e a r pUSVCAT, recombinant CAT phage, and CAT phage DNA) were detected. CAT expression signals varied i n strength, and are a r b i t r a r i l y denoted by '+' to '+++' symbols. The observation i n many instances that some F^ batches were positive while other batches from the same parent were negative indicate that the percentage of CAT-positive offspring was low, and that the germline-positive parents were probably mosaic for the CAT sequences. 40 Figure 1 1 Inherited CAT gene expression i n pooled offspring of pUSVCAT DNA-treated parents. F e r t i l i z e d eggs that had been injected with (a) supercoiled, and (b) l i n e a r , pUSVCAT DNA were reared to maturity and outcrossed with untreated f i s h . Offspring from each outcross were pooled into batches of 5 and assayed for the presence of CAT enzyme a c t i v i t y . Autoradiograms show representative CAT assays of pooled F^ from 5 different parents for each treatment group. Alpha-numerals at the bottom of each lane i d e n t i f y the different parents. See Table 1 and 2 for detailed results of F-^  CAT assays. 41 42 Figure 12 Inherited CAT gene expression i n pooled offspring of CAT phage p a r t i c l e and CAT phage DNA treated parents. F e r t i l i z e d eggs that had been injected with (a) recombinant CAT phage p a r t i c l e s , and (b) p u r i f i e d CAT phage DNA, were reared to maturity and outcrossed with untreated f i s h . Offspring were pooled and assayed for CAT a c t i v i t y , and autoradiograms of representative CAT assays shown, as described i n Fig 1 1 . See Table 3 and 4 for detailed results of F-L CAT assays. 4 4 Table 1. Inherited expression of supercoiled pUSVCAT DNA i n offspring. Medaka eggs injected with supercoiled pUSVCAT DNA were reared to adulthood and outcrossed with untreated f i s h . Offspring were pooled into batches of 5 and assayed for CAT enzyme a c t i v i t y , m, male; f , female; -, no detectible CAT a c t i v i t y ; + to +++, r e l a t i v e CAT a c t i v i t y strengths; nd, not done. 1 batch no. 2 3 Fo Sex SI m + + nd S2 m - - -S3 m - - -S4 m - + -S5 f + + -S6 f + + + S7 f + + -S8 f - +++ + S9 f + + -S10 f + + + S l l m - - nd S12 f - - nd S13 m + + nd S14 f + + -S15 f + + + S16 m - + -S17 m + + + S18 f - - + S19 f + + + S20 f - - -S21 f - - -S22 f - - -S23 f ++ - nd S24 f ++ ++ ++ 45 Table 2. Inherited expression of line a r pUSVCAT DNA i n off s p r i n g . Parents were derived from eggs injected with l i n e a r pUSVCAT DNA. Procedure for screening of offspring as described i n Table 1. F-^  batch no. *0-L l L2 L3 L4 L5 L6 L7 L8 L9 Sex m m m m f f f m +++ + + + nd nd +++ nd 46 Table 3. Inherited CAT expression i n offspring of parents derived from f e r t i l i z e d eggs injected with recombinant CAT phage p a r t i c l e s . Procedure for screening of offspring as described i n Table 1. F-^  batch no. FQ Sex PI f - - nd P2 f P3 m + +++ P4 m P5 m - + P6 f +++ + P7 m - - + P8 f ++ ++ ++ P9 m - - -P10 f - - + P l l f + ++ + 47 Table 4. Inherited CAT expression i n offspring of parents derived from f e r t i l i z e d eggs injected with p u r i f i e d CAT phage DNA. Procedure for screening of offspring as described i n Table 1. 1 F-^  batch no. 2 3 4 Fo Sex Dl f + + + + D2 f ++ ++ + + D3 f + ++ - -D4 f + + + + D5 f ++ ++ ++ nd 48 DISCUSSION The expression and fate of a CAT reporter gene after microinjection into the cytoplasm of f e r t i l i z e d medaka eggs (1-2 c e l l stage embryos) were monitored during embryonic development and up to the free-swimming f i s h stage. This gene was applied as supercoiled or li n e a r plasmid DNA, recombinant phage p a r t i c l e s , or p u r i f i e d phage DNA. The i n j e c t i o n of supercoiled or li n e a r pUSVCAT DNA resulted i n peak CAT gene expression at the gastrula/neurula stage (1 day po s t - i n j e c t i o n ) , followed by a sustained though s l i g h t l y weaker expression i n the one-week old embryo. Expression was s i g n i f i c a n t l y reduced, and varied noticeably, i n the hatchlings sampled (2 weeks post - i n j e c t i o n ) . By the free-swimming f i s h stage (4 weeks po s t - i n j e c t i o n ) , expression was detected i n only a few of the sampled f i s h . The r e l a t i v e l y s i m i l a r expression patterns obtained with supercoiled and li n e a r plasmid DNA indicate that physical conformation of input DNA has no s i g n i f i c a n t effect on i t s a b i l i t y to be expressed. These results contrast with two c o n f l i c t i n g observations i n the Xenopus embryo; Etkin and B a l c e l l s (1985) reported a higher expression for supercoiled pSV2CAT DNA and lower CAT a c t i v i t y i f the plasmid was f i r s t l i n e a r i z e d , whereas Wilson et al. (1986) observed that a cloned a c t i n gene and an actin-globin fusion gene were e f f i c i e n t l y transcribed only i f the c i r c u l a r plasmid containing either gene was f i r s t l i n e a r i z e d . The CAT expression pattern obtained when medaka eggs were injected with CAT phage DNA d i f f e r e d s i g n i f i c a n t l y from that obtained with pUSVCAT DNA; peak CAT gene expression did not occur u n t i l the one-week old embryonic stage. Expression characteristics at the hatchling and free-swimming f i s h stages, however, were si m i l a r to that observed with pUSVCAT i n j e c t i o n s . The increase i n expression from the gastrula/neurula stage to the one-week old embryo stage i s surprising since the amount of CAT phage DNA i s maximal at the 49 gastrula/neurula stage and i s s i g n i f i c a n t l y reduced thereafter. With CAT phage p a r t i c l e s , a sim i l a r pattern of increasing CAT a c t i v i t y up to the one-week old embryo stage and subsequent decline was observed. These observations suggest a possible role of the phage vector arms i n post-gastrula/neurula enhancement of gene expression. In an experiment involving the microinjection of a lambda DNA clone into f e r t i l i z e d Xenopus eggs, correctly l o c a l i z e d t r a n s c r i p t i o n was achieved when a mixture of the a c t i n gene insert and the vector arms was injected, but not when the intact clone was used (Wilson et al., 1986). This suggests that the lambda sequences may have a c i s - i n h i b i t o r y effect on tr a n s c r i p t i o n of the a c t i n gene i n the Xenopus embryo. Analysis of embryos at e a r l i e r stages revealed that the injected supercoiled or li n e a r pUSVCAT DNA was not expressed p r i o r to the early high b l a s t u l a stage (4 hr post-injection) although approximately 25 pg DNA, corresponding to 3 x 10^ copies of the CAT gene, was injected into each 1-2 c e l l stage embryo. The onset of CAT expression i n medaka embryos only after the early high b l a s t u l a stage appears to p a r a l l e l a phenomenon observed i n Xenopus embryos by Newport and Kirschner (1982a,b). These authors showed that t r a n s c r i p t i o n of endogenous or injected DNA i n Xenopus embryos begins at stage termed the 'mid-blastula t r a n s i t i o n ' (Gerhart, 1980), when a c r i t i c a l r a t i o between nucleus and cytoplasm i s reached. They also showed that t r a n s c r i p t i o n a l suppression of a yeast leucine tRNA gene which was injected at pre-mid-blastula stages could be reversed by i n j e c t i n g an amount of pBR322 DNA equivalent to the t o t a l genomic DNA that i s present i n a mid-blastula stage embryo, thereby t i t r a t i n g out presumed suppressor components. Etkin and B a l c e l l s (1985), using the pSV2CAT plasmid DNA, were also unable to detect expression of the CAT gene pr i o r to the mid-blastula t r a n s i t i o n . They argued against the p o s s i b i l i t y that the appearance of CAT a c t i v i t y only at the mid-bl a s t u l a t r a n s i t i o n 8 hr after i n j e c t i o n was simply a consequence of increase i n pSV2CAT DNA copy number after several rounds of r e p l i c a t i o n , since i n j e c t i o n of an equal amount into non-replicating oocytes e l i c i t e d CAT expression as early as 2.5 hr after i n j e c t i o n . Furthermore, i n j e c t i o n of a d i f f e r e n t CAT plasmid construct together with a trans-activating enhancing factor did not induce e a r l i e r expression, but did induce a much stronger expression at the mid-blastula t r a n s i t i o n . In the present study, i n j e c t i o n of higher DNA amounts equivalent to the genomic DNA content of the medaka mid-b l a s t u l a was not attempted because exogenous DNA doses above 250 pg have been shown to be l e t h a l to the medaka embryo (Vielkind et al. , 1988). Although CAT phage DNA injected eggs were not analysed at the stages e a r l i e r than the gastrula/neurula stage, the moderately strong signal seen at t h i s stage implies that the expression of the input CAT phage DNA also began an e a r l i e r embryonic stage. In marked contrast, a t r a n s c r i p t i o n i n i t i a t i o n stage similar to the Xenopus mid-blastula t r a n s i t i o n i s not present during sea urchin embryogenesis . For example, sea urchin early histone genes are expressed during early cleavage (reviewed i n Davidson, 1976) since the l i m i t e d histones present i n the comparatively smaller egg are s u f f i c i e n t only for a few cleavages (Poccia et a l . , 1981). Injected early histone H2A ( V i t e l l i et al.,1988) and early histone H2B (Colin et al., 1988) genes showed peak tr a n s c r i p t i o n together with endogenous genes during the early b l a s t u l a stage. As noted for a l l four treatment groups, for each of the stages up to the one-week old embryo stage, CAT expression signals were highly consistent among the samples analysed. At the hatchling stage, signal strengths among the samples varied noticeably, and by the free-swimming f i s h stage CAT expression could not be detected i n a s i g n i f i c a n t l y large f r a c t i o n of the samples. These observations are reflected i n the fate of the introduced plasmid and phage DNA sequences whereby, at embryonic stages up to the one-week old embryo stage, the amount of foreign DNA sequences present at each stage was f a i r l y consistent among the sampled embryos. Amounts of exogenous DNA increased during cleavage and gastrulation and were s i g n i f i c a n t l y reduced by the one-51 week old embryo stage. Thereafter, DNA amounts fluctuated among the sampled hatchlings, and was no longer detectible i n most of the sampled free-swimming f i s h . Upon i n j e c t i o n , supercoiled pUSVCAT DNA was gradually converted to a high molecular weight concatemer oriented head-to-tail. This high molecular weight form was observed to increase greatly during cleavage and gastrulation, suggesting that i t i s the preferred r e p l i c a t i v e structure. When linea r pUSVCAT DNA was injected, a very rapid conversion to high molecular weight concatemers occurred, r e s t r i c t i o n analysis of which revealed a random orientation of the li g a t e d molecules. Some conversion of the li n e a r molecules to supercoils, open c i r c l e s , and multimeric c i r c l e s was also evident and the high molecular weight form was also rapidly replicated during cleavage and gastrulation. I t i s highly improbable that the 'appearance' of the three c i r c u l a r forms resulted from r e p l i c a t i o n of trace amounts already present i n the stock DNA solution; some supercoils and open c i r c l e s were already detectible i n embryos analysed within 5 min of t h e i r i n j e c t i o n with lin e a r pUSVCAT (see Fig 6b, lane 1), and Southern bl o t hybridization of li n e a r pUSVCAT standards containing the equivalent of one to four times the injected amount showed no evidence of these c i r c u l a r forms (data not shown). The results obtained with supercoiled and li n e a r plasmid DNA are v i r t u a l l y i d e n t i c a l to the observations by Marini et al. (1988) i n the Xenopus embryo but d i f f e r somewhat from the processes occurring i n the sea urchin embryo. In the sea urchin embryo, supercoiled plasmids neither l i g a t e nor replicate and only l i n e a r molecules are rapidly and e f f i c i e n t l y assembled into end-to-end concatenates (McMahon et al., 1985). In addition, l i n e a r molecules injected into sea urchin embryos are not able to reform supercoils or c i r c u l a r molecules (McMahon et al., 1985). Marini et al. (1988) noted that the high molecular weight concatemer formed i n Xenopus embryos from l i n e a r plasmid molecules may e x i s t as a random concatemer or may exhibit a preference for head-to-tail orientation. A sim i l a r mechanism may e x i s t i n the medaka embryo, 52 which could explain why r e s t i c t i o n analysis of the l i n e a r plasmid-derived concatemers yielded more of the fragment expected from both un i d i r e c t i o n a l and random l i g a t i o n s and less of the fragments only expected from random l i g a t i o n s . The formation of random concatemers generated by l i n e a r molecules i s presumably due to random l i g a t i o n of l i n e a r termini. A Xenopus ovary s p e c i f i c protein that promotes concatenation i n v i t r o of l i n e a r DNA (Bayne et a.1., 1984) and a sea urchin early embryo s p e c i f i c DNA ligase that acts on l i n e a r DNA with cohesive or blunt ends (Prigent et al., 1987) have been is o l a t e d . Presumably a s i m i l a r a c t i v i t y i s present i n medaka embryos to assemble long concatemers from unit length l i n e a r DNA. The exclusively head-to - t a i l orientation of concatemers generated by supercoiled DNA indicate that, apart from random l i g a t i o n , another mechanism related to homologous recombination possibly exists i n the medaka embryo, as postulated for Xenopus embryos (Marini et al., 1988). Folger et al. (1982) have shown i n mammalian c e l l s that injected l i n e a r or supercoiled plasmids are organized into tandem head-to-tail arrays through homologous recombination between plasmid molecules. The presence of high molecular weight forms after i n j e c t i o n of l i n e a r plasmid DNA has been reported i n embryos of other f i s h species (Zhu et al., 1986; Dunham et al., 1987; Stuart et al., 1988). No rapid conversions of the sort observed with plasmid DNA injections occurred when medaka eggs were injected with either CAT phage p a r t i c l e s or CAT phage DNA. Instead, an upward s h i f t consistent with a change into higher molecular weight structures was obvious only from the one-week old embryonic stage onwards. Heating of the embryo DNA extracts at 65 °C followed by rapid cooling p r i o r to gel electrophoresis and Southern b l o t analysis did not produce a band corresponding to CAT phage DNA monomers. I t i s possible that the l i n e a r phage molecules were covalently l i g a t e d into concatemers either with or without modification of the i r cohesive ends. Covalent end-to-end 53 l i g a t i o n o f a r e c o m b i n a n t A c l o n e ha s b e en o b s e r v e d a f t e r p r o n u c l e a r i n j e c t i o n i n t o f e r t i l i z e d mouse eggs ( C o s t a n t i n i and L a c y , 1 9 8 1 ) . A c o m p a r i s o n o f t h e e x t e n t o f r e p l i c a t i o n be tween s u p e r c o i l e d and l i n e a r p l a s m i d DNA i n j e c t e d embryos p o i n t s t o a s l i g h t l y g r e a t e r deg r ee o f p l a s m i d DNA r e p l i c a t i o n when t he l i n e a r m o l e c u l e i s u s e d . T h i s m i g h t be e x p e c t e d c o n s i d e r i n g t h e much q u i c k e r c o n v e r s i o n o f i n p u t l i n e a r m o l e c u l e s i n t o h i g h m o l e c u l a r w e i g h t c o n c a t e m e r s , w h i c h appea r t o be t h e p r e f e r r e d r e p l i c a t i v e f o r m . F o r eggs i n j e c t e d w i t h CAT phage p a r t i c l e s , t o t a l CAT DNA was r e l a t i v e l y unchanged up t o t h e e a r l y h i g h b l a s t u l a (4 h r p o s t - i n j e c t i o n ) , p o s s i b l y s u g g e s t i n g t h a t most o f t h e phage DNA ha s y e t t o be r e l e a s e d f r om the phage c o a t s . The i n c r e a s e d t o t a l CAT DNA a t t h e f l a t b l a s t u l a s t a g e (10 h r p o s t - i n j e c t i o n ) m i g h t be e x p l a i n e d b y a s ub sequen t r e l e a s e o f DNA f r om the phage c o a t s and e n s u i n g DNA r e p l i c a t i o n . The r e a ppea r s t o be no f u r t h e r i n c r e a s e s i n DNA a f t e r t h i s s t a g e , amounts r e m a i n i n g unchanged even a t t he 30 s o m i t e n e u r u l a s t a g e (72 h r p o s t - i n j e c t i o n ) . The l a c k o f a p r o t e c t i v e phage c o a t a r o u n d t h e i n j e c t e d p u r i f i e d CAT phage DNA p r o b a b l y a c c o u n t s f o r t h e s i g n i f i c a n t l y g r e a t e r deg r ee o f DNA r e p l i c a t i o n and d e g r a d a t i o n as compared t o CAT phage p a r t i c l e - i n j e c t e d embryos . The i n p u t CAT phage DNA was , howeve r , n o t as e x t e n s i v e l y r e p l i c a t e d as i n p u t p l a s m i d DNA o f s u p e r c o i l e d o r l i n e a r c o n f o r m a t i o n . By t h e 30 s o m i t e n e u r u l a s t a g e , embryos d e r i v e d f r om p l a s m i d and phage DNA i n j e c t i o n s c o n t a i n e d s i g n i f i c a n t l y r e d u c e d exogenous DNA. S i m i l a r o b s e r v a t i o n s o f r e p l i c a t i o n up t o t h e g a s t r u l a / n e u r u l a s t a g e and s ub s equen t l o s s o f exogenous s u p e r c o i l e d and l i n e a r p l a s m i d DNA ha s b e en o b s e r v e d i n Xenopus ( R u s c o n i and S c h a f f n e r , 1 9 8 1 ) . I n s e a u r c h i n s (McMahon e t al., 1985) and f i s h (Zhu e t al., 1 9 8 6 ) , a s i m i l a r s t a g e s p e c i f i c i n c r e a s e and d e c r e a s e o f i n j e c t e d l i n e a r p l a s m i d DNA ha s been r e p o r t e d . S i n c e t h e m a j o r i t y o f i n p u t DNA sequences t h a t were r e p l i c a t e d were c o n t i n u a l l y b e i n g d eg r aded and s u b s e q u e n t l y l o s t , i t i s l i k e l y t h a t t h e y r e m a i n e d e x t r a - c h r o m o s o m a l t h r o u g h o u t e m b r y o g e n e s i s . P r e s u m a b l y , e x t r a -54 chromosomally replicated foreign DNA i s unequally segregated among the daughter c e l l s of a rapidly dividing and growing embryo, r e s u l t i n g i n some of the c e l l s not having the exogenous sequences at a l l . In addition, exogenous DNA segregated into a c t i v e l y dividing c e l l s of rapidly growing tissues would continue to be replicated, whilst those ending up i n non-dividing or slowly d i v i d i n g c e l l s of d i f f e r e n t i a t e d tissues would be quickly degraded and l o s t . The net r e s u l t would be an animal mosaic for the presence of the foreign DNA. Mosaicism has been demonstrated i n Xenopus where pSV2CAT DNA injected into f e r t i l i z e d eggs persisted i n tissues of adult frogs but exhibited a mosaic pattern of d i s t r i b u t i o n (Etkin and Pearman, 1987). Hough-Evans et al. (1988) have also shown by direct in situ DNA hybridization that exogenous CAT DNA sequences injected into f e r t i l i z e d sea urchin eggs are mosaically distributed to most or a l l c e l l types or lineages of the embryo. This unequal d i s t r i b u t i o n and resultant mosaicism of non-chromosomal exogenous DNA may explain the greater CAT expression variations among the medaka hatchlings and loss of CAT enzyme a c t i v i t y i n many free-swimming f i s h . Despite the fact that most of the input and replicated DNA remain extra-chromosomal, the p o s s i b i l i t y exists that genomic integration of some of the exogenous sequences occurred. In t h i s present study, integration of the injected CAT plasmid and phage sequences into the medaka genome i s supported by the persistence of CAT DNA sequences and of CAT gene expression i n a few free-swimming f i s h , and by the demonstration of inherited CAT gene expression i n pooled offspring of several DNA and phage-treated f i s h . Since germline-positive parents were i d e n t i f i e d from a l l four treatment groups, i t would appear that neither the i n i t i a l DNA conformation, nor d i f f e r e n t vector sequences adversely affect the a b i l i t y of a gene to be inherited by offspring. Neither does packaging of DNA into phage p a r t i c l e s appear to be a b a r r i e r to germline transmission of the enclosed gene. The observation that some F-^  pools were p o s i t i v e , while others from the same parent were not, suggests that 55 the germline-positive parents were mos>aic for the integrated sequences. This would be expected i f the assumption i s made that the incorporation of the foreign sequences did not occur immediately after i n j e c t i o n , and that the exogenous DNA was incorporated into only one or a few of several primordial germ c e l l s . In the medaka, Gamo (1961) determined that no primordial germ c e l l s could be distinguished p r i o r to gastrulation, but that up to 20 such c e l l s could be determined at the early gastrula stage (15 hr post-f e r t i l i z a t i o n , the equivalent of 13 hr p o s t - i n j e c t i o n ) . Genomic integration at an early cleavage stage would t h e o r e t i c a l l y r e s u l t i n a higher probability that a l l the primordial germ c e l l s contain the integrated foreign sequence. The observation that some of the tested f i s h were not germline positive does not exclude the p o s s i b i l i t y that they may harbour exogenous sequences i n a f r a c t i o n of t h e i r c e l l s i n other tissues. Genomic integration of exogenous DNA sequences after i n j e c t i o n into the cytoplasm of f e r t i l i z e d sea urchin egg has been demonstrated by Flytzanis et a l . (1985). In addition, germline transmission of cloned genes after microinjection into the egg cytoplasm has recently been reported i n Xenopus (Etkin and Pearman, 1987) and i n f i s h (Guyomard et a l . , 1988; Stuart et a l . , 1988; V i e l k i n d et a l . , 1988). In a l l the above cases, the animals were mosaic for the exogenous sequences, and mosaicism of the founder animals was inferred from the observation that only a percentage of the offspring from each founder were positive for the presence of the exogenous sequences. I t should be noted that germline transmission p e r se does not prove stable host genome integration, as was shown i n the nematode i n which an episomal structure was passed to the offspring (Stinchcomb et a l . , 1985). Thus, genomic integration has to be further substantiated by analysis of Mendelian inheritance of the transgene. Stuart et a l . (1988) were able to demonstrate stable integration of a l i n e a r plasmid i n the zebrafish genome, showing that 50% of F2 progeny issued from a foreign DNA positive F-^  outcrossed to an untreated f i s h carried the foreign sequence. 56 The CAT assay procedure adopted i n t h i s study was devised as a quick screen to enable a large number of parents to be rapidly tested for germline transmission of functional CAT sequences. Dot b l o t or s l o t b l o t analysis has been employed i n zebrafish (Stuart et a l . , 1988) and Southern bl o t analysis used i n trout (Guyomard et a l . , 1988) to test offspring for the presence of foreign DNA sequences. However, DNA loading constraints for dot/slot blots dictate the need to load equal amounts of DNA within a narrow range. A minimum amount of t o t a l DNA i s required i n order for single copy genes to be detected. Offspring cannot be pooled since excess t o t a l DNA may compete out any weak hybridization signal or conversely resul t i n high background signals. The DNA extraction and hybridization procedures are also comparatively elaborate. Southern bl o t analysis allows more DNA to be loaded and thus could allow pooling of offspring from a single parent, but i s comparatively slower than either dot/slot b l o t analysis or CAT enzyme assay. Most importantly, a p o s i t i v e signal from DNA analysis provides no information on whether the transmitted CAT gene i s functional. Using the simpler and quicker CAT assay protocol, tests with pooled embryos or hatchlings from non-treated parents were negative for CAT a c t i v i t y , and no i n h i b i t o r y effect could be detected when a commercial CAT enzyme was incubated i n pooled untreated embryo/hatchling extracts. Thus, not only could F-^  embryos and hatchlings from the same DNA/phage treated parent be pooled, but a positive CAT signal indicated germline transmission of f u l l y functional CAT sequences. Taken together, the results of t h i s study indicate that cloned DNA and recombinant phage p a r t i c l e s , cytoplasmically injected at the 1-2 c e l l stage of medaka embryos, pe r s i s t and are expressed during embryogenesis. No s i g n i f i c a n t advantage with regard to expression and persistence was observed when either l i n e a r or supercoiled plasmid molecules were used, but an apparently more e f f i c i e n t expression was observed with the plasmid DNA clone than with the phage DNA clone. Nonetheless, the successful expression and 57 r e p l i c a t i o n of input phage DNA clone opens the p o s s i b i l i t y of testing larger genes, especially genomic sequences containing long stretches of introns or distant regulatory regions, which cannot be cloned into plasmids. In addition, providing s u f f i c i e n t l y high phage p a r t i c l e t i t r e s are used, genes cloned into recombinant phages can be tested by d i r e c t i n j e c t i o n of phage p a r t i c l e s without having to f i r s t extract the DNA. The very consistent CAT expression signals i n the early embryonic stage of the medaka strongly favour i t s use as a transient expression system i n the analysis of 'early' gene regulatory regions, as has been done, e.g. i n sea urchins ( F l y t z a n i s , et a l . , 1987). In addition, the medaka embryo can be use i n the functional testing of genes intended to be used i n the genetic engineering of economically important f i s h e s . For example, genes coding for the winter flounder antifreeze proteins (Lin and Gross, 1981; Davies et a l . , 1982; Gourlie et a l . , 1984), salmon beta-gonadotropin (Trinh et a l . , 1986), and rainbow trout growth hormone (Agellon et a l . , 1988) have been cloned. Introduction of such genes into the genome of f i s h could p o t e n t i a l l y a l t e r t h e i r cold water tolerance, f e r t i l i t y , and growth rate, respectively. The a b i l i t y of the exogenous DNA sequences to p e r s i s t i n some free-swimming stage medaka, and the positive F-^  CAT assay r e s u l t s , also open the p o s s i b i l i t y of creating transgenic l i n e s of medaka for the study of c e l l lineages and ' l a t e ' gene regulation, among others. The embryo i s well suited for these purposes since the r e l a t i v e l y large embryo size (ca 1.5 mm diameter) makes manipulation under low magnifications (8x to 50x) possible, the two c e l l s derived from f i r s t cleavage are very well defined, and sharp i n j e c t i o n needles can e a s i l y penetrate the chorion. Although not employed i n these studies, the chorion can be made more penetrable by mild digestion with proteinase K or pronase without having any effect on the v i a b i l i t y of the embryos. Thus, harsh treatments such as trypsin-urea used to dissolve the chorion of goldfish eggs (Yamaha et a l . , 58 1986) or manipulations such as boring ti n y holes into the chorion of trout eggs p r i o r to i n j e c t i o n (Rokkones et a l . , 1985) are unneccessary. This technique also obviates the more d i f f i c u l t task of i n j e c t i n g into the germinal v e s i c l e of the medaka oocyte, a procedure adopted by Ozato et a l . (1986), since injections into the cytoplasm are s u f f i c i e n t to e l i c i t gene expression and germline transmission of the foreign sequences. REFERENCES 59 Agellon, L.B., Davies, S.L., Chen, T.T., and Powers, D.A. (1988). Structure of a fis h (rainbow trout) growth hormone gene and i t s evolutionary implications. Proc. Natl. Acad. Sci. U.S.A. 8 5 , 5136-5140. Bayne, M.L., Alexander, R.F., and Benbow, R.M. (1984). DNA binding protein from ovaries of the frog Xenopus laevis which promotes concatenation of linear DNA. J . Mol. B i o l . 1 7 2 , 87-108. Bendig, M.M., and Williams, J.G. (1983). Replication and expression of Xenopus laevis globin genes injected into f e r t i l i z e d Xenopus eggs. Proc. Natl. Acad. Sci. U.S.A. 8 0 , 6197-6201. Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K., and Palmiter, R.D. (1985) . Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. U.S.A. 8 2 , 4438-4442. Chourrout, D., Guyomard, R., and Houdebine, L-M. (1986). High efficiency gene transfer in rainbow trout (Salmo gairdneri Rich.) by microinjection into egg cytoplasm. Aquaculture 5 1 , 143-150. Clewell, D.B. and Helinski, D.R. (1969). Supercoiled circular protein complex in Escherischia c o l i , purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 6 2 , 1159-1166. Colin, A.M., Catlin, T.L., Kidson, S.H., and Maxson, R. (1988). Closely linked early and late histone H2B genes are diff e r e n t i a l l y expressed after microinjection into sea urchin zygotes. Proc. Natl. Acad. Sci. U.S.A. 8 5 , 507-510. Costantini, F. and Lacy, E. (1981). Introduction of a rabbit ^-globin gene into the mouse germ li n e . Nature 2 9 4 , 92-94. Crabb, D.W. and Dixon, J.E. (1987). A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured c e l l s . Anal. Biochem. 1 6 3 , 88-92. Crittenden, L.B. and Salter, D.W. (1985). Genetic engineering to improve resistence to v i r a l diseases of poultry: A model for application to livestock improvement. Can. J . Anim. Sci. 6 5 , 553-562. Davidson, E.H. (1976). Gene activity in early development. Academic Press, New York. Davidson, E.H., Flytzanis, C.N., Lee, J.J., Robinson, J.J., Rose, S.J., and Sucov, H.M. (1985). Lineage-specific gene expression in the sea urchin embryo. Cold Spring Harbour Symp. Quant. B i o l . 5 0 , 321-328. Davies, P.L., Roach, A.H., and Hew, C-L. (1982). DNA sequence coding for an antifreeze protein precursor from winter flounder. Proc. Natl. Acad. Sci. U.S.A. 79, 335-339. 60 Dunham, R.A., Eash, J . , Askins, J . , and Townes, T.M. (1987). Transfer of the metallothionein-human growth hormone fusion gene into channel catfish. Trans. Am. Fish. Soc. 116, 87-91. Etkin, L.D. (1982). Analysis of the mechanisms involved in gene regulation and c e l l differentiation by microinjection of purified genes and somatic c e l l nuclei into amphibian oocytes and eggs (review). Differentiation 21, 149-159. Etkin, L.D., and Balcells, S. (1985). Transformed Xenopus embryos as a transient expression system to analyze gene expression at the midblastula transition. Dev. B i o l . 108, 173-178. Etkin, L.D. and Pearman, B. (1987). Distribution, expression and germline transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs. Development 99, 15-23. Etkin, L.D., Pearman, B., Roberts, M., and Bektesh, S.L. (1984). Replication, integration, and expression of exogenous DNA injected into f e r t i l i z e d eggs of Xenopus laevis. Differentiation 36, 194-202. Feinberg, A.P. and Vogelstein, B. (1983/84). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13 and 137, 266-267. Fire, A. (1986). Integrative transformation of Caenorhabditis elegans. EMBO J . 5, 2673-2680. Flytzanis, C.N., McMahon, A.P., Hough-Evans, B.R., Katula, K.S., Britten, R.J., and Davidson, E.H.(1985). Persistence and integration of cloned DNA in postembryonic sea urchins. Dev. Bi o l . 108, 431-442. Flytzanis, C.N., Britten, R.J.,and Davidson, E.H. (1987). Ontogenic activation of a fusion gene introduced into the sea urchin egg. Proc. Natl. Acad. Sci. U.S.A. 84, 151-155. Folger, K.R., Wong, E.A., Wahl, G., and Capecchi, M.R. (1982). Patterns of integration of DNA microinjected into cultured mammalian c e l l s : Evidence for homologous recombination between injected plasmid DNA molecules. Mol. Cell B i o l . 2, 1372-1387. Franks, R.R., Hough-Evans, B.R., Britten, R.J., and Davidson, E.H. (1988). Spatially deranged though temporally correct expression of a Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family. Genes & Development 2, 1-12. Gamo, H. (1961). On the origin of germ cells and formation of gonad primordia in the medaka, Orvzias latipes. Japan J . Zool. 13, 101-115. Gerhart, J.G. (1980). Mechanisms regulating pattern formation in the amphibian egg and early embryo. In: Biological regulation and development, 2, R.F. Goldberger, ed. Plenum Press, New York, pp. 133-315. Gorman, CM., Moffat, L.F., and Howard, B.H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian c e l l s . Mol. C e l l . B i o l . 2, 1044-1051. 61 Gourlie, B., Lin, Y., Price, J . , De Vries, A.L., Powers, D., and Huang, R.C.C. (1984). Winter flounder antifreeze proteins: A multigene family. J . Bi o l . Chem. 259, 14960-14965. Guyomard, R., Chourrout, D., and Houdebine, L. (1988). Production of stable transgenic fi s h by cytoplasmic injection of purified genes. J . C e l l . Biochem. (in press). Hammer, R.E., Pursel, V.G., Rexroad, C.E.Jr., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D., and Brinster, R.L. (1985). Production of transgenic rabbits, sheep, and pigs by microinjection. Nature 315, 680-683. Hew, C , Davies, P.L., Shears, M. , King, M. , and Fletcher, G. (1987). Antifreeze protein gene transfer to atlantic salmon by microinjection (abstract). Federation Proc. 46, 2039. Hough-Evans, B.R., Franks, R.R., Cameron, R.A., Britten, R.J., and Davidson, E.H. (1987). Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs. Dev. Bi o l . 121, 576-579. Hough-Evans, B.R., Britten, R.J., and Davidson, E.H. (1988). Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo. Dev. Bio l . 129, 198-208. Ishiura, M., Hirose, S. Uchida, T., Hamada, Y., Suzuki, Y., and Okada, Y. (1982). Phage particle-mediated gene transfer to cultured mammalian c e l l s . Mol. C e l l . B i o l . 2, 607-616. Jaenisch, R. (1988). Transgenic Animals. Science 240, 1468-1474. Karlsson, S., Humphries, R.K., Gluzman, Y., and Nienhuis, A.W. (1985). Transfer of genes into hematopoietic cells using recombinant DNA viruses. Proc. Natl. Acad. Sci. U.S.A. 82, 158-162. Katula, K.S., Hough-Evans, B.R., Britten, R.J., and Davidson, E.H. (1987). Ontogenic expression of a Cyl:actin fusion gene injected into sea urchin eggs. Development 101, 437-447. King, D. and Wall, R.J. (1988). Identification of specific gene sequences in preimplantation embryos -- detection of a transgenome (abstract). J . C e l l . Biochem. Suppl. 12B, 190. Kirchen, R.V. and West, W.R. (1976). The Japanese medaka -- i t s care and development. Carolina Biological Supply Company (publ.) 36pp. Krieg, P.A., and Melton, D.A. (1985). Developmental regulation of a gastrula-specific gene injected into f e r t i l i s e d Xenopus eggs.. EMBO J . 4, 3463-3471. Lin, Y., and Gross, J.K. (1981). Molecular cloning and characterization of winter flounder antifreeze cDNA. Proc. Natl. Acad. Sci. U.S.A. 78, 2825-2829. Maclean, N., Penman, D., and Zhu, Z. (1987). Introduction of novel genes into f i s h (review). Bio/Technology 5, 257-261. 62 Maniatis, T., Frisch, E.F. and Sambrook, J . (1982). Molecular Cloning: Laboratory Manual. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory. Marini, N.J., Etkin, L.D., and Benbow, R.M. (1988). Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis. Dev. B i o l . 127, 421-434. Matsui, K. (1949). Illustration of the normal course of development in the Oryzias latipes (in Japanese). Jap. J . Exp. Morphol. 5, 33-42. McMahon, A.P., Flytzanis, C.N., Hough-Evans, B.R., Katula, K.S., Britten, R.J., and Davidson, E.H. (1985). Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev. B i o l . 108, 420-430. McEvoy, T.G, Stack, M., Barry, T., Keane, B., Gannon, F., and Sreenan, J.M. (1987). Direct gene transfer by microinjection (abstract). Theriogenology 27, 258. Nancarrow, C , Marshall, J . , Murray, J . , Hazelton, I., and Ward, K. (1987). Production of a sheep transgenic with the ovine growth hormone gene (abstract). Theriogenology 27, 263. Newport, J . and Kirschner, M. (1982a). A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675-686. Newport, J . and Kirschner, M. (1982b). A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687-696. Okayama, H. and Berg, P. (1985). Bacteriophage lambda vector for transducing a cDNA clone library into mammalian c e l l s . Mol. C e l l . B i o l . 5, 1136-1142. Ozato, K., Kondoh, H., Inohara, H., Iwamatsu, T., Wakamatsu, Y., and Okada, T.S. (1986). Production of transgenic fish: Introduction and expression of chicken 5-crystallin gene in medaka embryos. Cell Differ. 19, 237-244. Palmiter, R.D. and Brinster, R.L. (1986). Germline transformation of mice. Ann. Rev. Genet. 20, 465-499. Poccia, D., Salik, J . , andKrystal, G. (1981). Transitions i n histone variant of the male pronucleus following f e r t i l i z a t i o n and evidence for a maternal store of cleavage-stage histones in the sea-urchin egg. Dev. B i o l . 82, 287-296. Prigent, C., Maniey, D., Lefresne, J . , Epel, D., Signoret, J . , and David, J-C (1987). Changes in the catalytic properties of DNA ligase during early sea urchin development. Dev. B i o l . 124, 281-286. Rokkones, E., Alestrom, P., Skjervold, H., and Gautvik, K.M. (1985). Development of a technique for microinjection of DNA into salmonid eggs. Acta Physiol. Scand. 124 suppl. 542, 417. 63 Rubin, G.M. and Spradling, A.C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science 218, 348-353. Rusconi, S. and Schaffner, W. (1981). Transformation of frog embryos with a rabbit 0-globin gene. Proc. Natl. Acad. Sci. U.S.A. 78, 5051-5055. Sleigh, M.J. (1986). A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic c e l l s . Anal. Biochem. 156, 251-256. Spradling, A.C. and Rubin, G.M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341-347. Spradling, A.C. and Rubin, G.M. (1983). The effect of chromosomal position on the expression of the Drosophila germ line chromosomes. Cell 34, 47-57. Stinchcomb, D.T., Shaw, J.E., Carr, S.H., and Hirsh, D. (1985). Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. C e l l . B i o l . 5, 3484-3496. Stuart, G.W., McMurray, J.V., and Westerfield, M. (1988). Replication, integration, and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103, 403-412. Trinh, K-Y. , Wang, N.C., Hew, C L . , and Crim, L.W. (1986). Molecular cloning and sequencing of salmon gonadotropin f) subunit. Eur. J . Biochem. 159, 619-624. Uwa, H. and Iwata, A. (1981). Karyotype and cellular DNA content of Oryzias  1avanicus (Oryziatidae, Pisces). Chromosome Inf. Serv. 31, 24-26. Vielkind, J.R. and Vogel, K.S. (1988). Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes. Pigment Cell Res. 2, 00. Vielkind, J.R., Chong, S.S.C, Stuart, G.W. , and Sadaghiani, B. (1988). Transgenic medaka and zebrafish systems: Transient and inherited expression of a recombinant CAT reporter gene microinjected into f e r t i l i z e d eggs. In: New Trends in Ichthyology, J.H. Schroeder, ed. Paul Parey, Munich (in press). V i t e l l i , L. , Kemler, I., Lauber, B., B i r n s t i e l , M.L., and Busslinger, M. (1988). Developmental regulation of micro-injected histone genes in sea urchin embryos. Dev. B i o l . 127, 54-63. Wilson, C , Cross, G.S., and Woodland, H.R. (1986). Tissue-specific expression of actin genes injected into Xenopus embryos. Cell 47, 589-599. Yamaha, E., Usui, K., Onozato, H., and Hamada, K. (1986). A method for dechorionation in goldfish, Carassius auratus. Bu l l . Jap. Soc. Scient. Fish. 52, 1929-1934. Yamamoto, T. (1961). Physiology of f e r t i l i z a t i o n in fi s h eggs. Int. Rev. Cytol. 12, 361-405. 64 Yamamoto, T. (1967). Medaka. In: Methods in developmental biology, Wilt, F.M. and Wessells, N.K., eds. T.Y. Growell, N.Y. (publ.) pp. 101-111. Yamamoto, T. (1975). Medaka ( k i l l i f i s h ) . Biology and strains. Keigaku Publishing Company, Tokyo. Zhu, Z., L i , G., He, L., and Chen, S. (1985). Novel gene transfer into the f e r t i l i z e d eggs of goldfish (Carassius auratus L 1758). J . Applied Ichthyology 1, 31-33. Zhu, Z., Xu, K., L i , G., Xie, Y., and He, L. (1986). Biological effects of human growth hormone gene microinjected into the f e r t i l i z e d eggs of loach Misgurnus anguillicaudatus (Cantor). Kexue Tongbao Academia Sinica 31, 988-990. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097426/manifest

Comment

Related Items