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

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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 l a t i p e s (TEMMINCK & SCHLEGEL) By SAMUEL SIONG CHUAN CHONG B . S c , The National U n i v e r s i t y of Singapore, SINGAPORE, 1985 B.Sc, (Hons), The National U n i v e r s i t y of Singapore, SINGAPORE, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1988 © Samuel Siong Chuan Chong, 1988  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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)  ii ABSTRACT  The c r e a t i o n o f mechanisms o f  gene r e g u l a t i o n ,  improvement o f In this 'medaka'  'transgenic'  livestock, thesis,  (Oryzias  latipes)  promoter-enhancer  as a phage,  cell  stage.  into  of  a supercoiled or l i n e a r  o r a s p u r i f i e d p h a g e DNA.  of the  analyses,  t e s t e d b y CAT enzyme a s s a y f o r  s l i g h t l y weaker  fish  The  reporter  plasmid  DNA o r p h a g e was  i n j e c t e d eggs  o f f s p r i n g were p o o l e d  t h e CAT  able  at  from the  the  in  t h e CAT gene up t o t h e  i n these treatments,  the s t r o n g e s t  early  late  varying noticeably detectible  the  but  significantly  R e c o m b i n a n t CAT p h a g e p a r t i c l e s  to express  and  pUSVCAT  a n d was s u s t a i n e d  E x p r e s s i o n was  CAT e x p r e s s i o n was s t i l l  were  sequences.  was s t r o n g e s t  (2 w e e k s p o s t - i n j e c t i o n ) ,  analysed.  monitored  t h a t DNA c o n f o r m a t i o n d o e s n o t a f f e c t  i n the one-week o l d embryo.  However,  of  1-2  a n d p e r f o r m i n g CAT enzyme  Several  (1 d a y p o s t - i n j e c t i o n ) ,  (4 w e e k s p o s t - i n j e c t i o n ) .  stage.  recombinant  CAT enzyme a c t i v i t y was d e t e c t i b l e  neurula stage  CAT p h a g e DNA w e r e a l s o fish  This  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  (4 h r p o s t - i n j e c t i o n ) ,  reduced i n h a t c h l i n g s individuals  and t h e i r  inheritance  indicating  of expression.  gastrula/early  or  gene r e g u l a t e d b y a d o u b l e  stages  respectively.  of expression of  DNA w e r e v e r y s i m i l a r ,  stage  fish  i n j e c t e d DNA o r p h a g e w e r e  developmental  allowed to develop to sexual maturity,  high blastula  genetic  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  and S o u t h e r n b l o t  efficiency  (CAT)  r e g i o n was c h o s e n a s a r e p o r t e r .  E x p r e s s i o n and f a t e  The p a t t e r n s  the Japanese r i c e f i e l d  acetyltransferase  b y h a r v e s t i n g medaka a t v a r i o u s assays  a s o p e n e d up a new a v e n u e f o r  a s a g e n e 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 .  eucaryotic  was i n t r o d u c e d a s e i t h e r  into  fish.  the s u i t a b i l i t y  chloramphenicol  microinjected  as w e l l  including  procaryotic  (pUSVCAT),  animals has p r o v i d e d i n s i g h t s  among t h e  free-swimming or  purified  free-swimming  CAT e x p r e s s i o n was  iii seen i n the one-week o l d embryo instead of i n the g a s t r u l a / n e u r u l a , r a i s i n g the p o s s i b i l i t y of a r o l e played by d i f f e r e n t vector sequences on gene expression. Studies on the f a t e of i n j e c t e d supercoiled and l i n e a r pUSVCAT revealed conversion of the input forms to high molecular weight h e a d - t o - t a i l and randomly o r i e n t e d concatemers r e s p e c t i v e l y .  T o t a l plasmid DNA increased  r a p i d l y during cleavage and g a s t r u l a t i o n , i n d i c a t i v e of plasmid r e p l i c a t i o n , whereas degradation of plasmid sequences was observed by the e a r l y 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 t e n - f o l d , whereas i n j e c t i o n of l i n e a r pUSVCAT r e s u l t e d 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 a f t e r the gastrula/neurula  stage, and t h i s DNA was e x c l u s i v e l y 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 i n j e c t e d CAT phage p a r t i c l e s nor the i n j e c t e d p u r i f i e d CAT phage DNA appeared to be concatenated during e a r l y embryogenesis. In both cases, however, the phage DNA appeared as higher molecular weight DNA by the one-week o l d embryonic stage, probably formed by covalent end-to-end ligations.  DNA of CAT phage p a r t i c l e s d i d not increase u n t i l a f t e r the e a r l y  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 p o s t - i n j e c t i o n ) a t h r e e - f o l d 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 t h e r e a f t e r . Injected p u r i f i e d CAT phage DNA increased through the stages of cleavage and g a s t r u l a t i o n , the gastrula/neurula having seven-fold more CAT phage DNA than that i n j e c t e d , and decreased t h e r e a f t e r . Both DNA of i n j e c t e d phage p a r t i c l e s and i n j e c t e d phage DNA could p e r s i s t to the freeswimming stage.  iv CAT gene expression was detected i n a number of pooled o f f s p r i n g from several DNA and phage-treated f i s h , i n d i c a t i n g inheritance of the input sequences.  The data i n t h i s study suggest that the germline-positive parents  are probably mosaic f o r the presence of the CAT sequences, and that germline transmission i s p o s s i b l e w i t h 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 w i t h the ease of handling and manipulation of the medaka embryo, strongly favour the use of the medaka as a t r a n s i e n t expression and transgenic animal model.  V  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  ACKNOWLEDGEMENTS  viii  INTRODUCTION  1  MATERIALS AND METHODS  6  RESULTS 1.  Studies of CAT reporter gene expression during medaka development. 1.1  1.2  1.3 2.  2.2  2.3  3.  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.  12  CAT gene expression due to cytoplasmic i n j e c t i o n o f 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  Onset o f CAT gene expression i n the e a r l y medaka embryo.  18  Studies on the fate o f introduced CAT DNA sequences during medaka development. 2.1  12 >  18  Conformational changes 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 plasmid DNA.  18  2.1.1 Nature of high molecular weight pUSVCAT form.  27  Conformational changes o f DNA introduced w i t h i n recombinant phage p a r t i c l e s and o f p u r i f i e d phage DNA.  30  R e p l i c a t i o n of i n j e c t e d f o r e i g n DNA sequences i n e a r l y embryos.  33  I n h e r i t e d expression of CAT reporter gene i n o f f s p r i n g .  36  DISCUSSION  48  REFERENCES  59  vi  LIST OF TABLES Page Table 1 Table 2 Table 3  Table 4  I n h e r i t e d expression of supercoiled pUSVCAT DNA i n o f f s p r i n g .  44  I n h e r i t e d expression of l i n e a r pUSVCAT DNA i n offspring.  45  I n h e r i t e d CAT expression i n o f f s p r i n g of parents derived from f e r t i l i z e d eggs i n j e c t e d with recombinant CAT phage p a r t i c l e s .  46  I n h e r i t e d CAT expression i n o f f s p r i n g of parents derived from f e r t i l i z e d eggs i n j e c t e d w i t h 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 l i n 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 a f t e r i n j e c t i o n of supercoiled and l i n e a r pUSVCAT DNA. Fate of i n j e c t e d supercoiled and l i n e a r pUSVCAT DNA during medaka development. E a r l y fate of i n j e c t e d supercoiled and l i n e a r pUSVCAT molecules. R e s t r i c t i o n fragment a n a l y s i s of high molecular weight pUSVCAT DNA. Fate of CAT phage DNA during medaka development a f t e r 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. R e p l i c a t i o n and subsequent decrease of input pUSVCAT DNA during e a r l y development. R e p l i c a t i o n and subsequent decrease of input CAT phage DNA during e a r l y development. I n h e r i t e d CAT gene expression i n o f f s p r i n g of pUSVCAT DNA-treated parents. I n h e r i t e d CAT gene expression i n o f f s p r i n g of parents t r e a t e d with CAT phage p a r t i c l e s and p u r i f i e d CAT phage DNA.  viii  ACKNOWLEDGEMENTS  I wish t o express my sincere appreciation: to my s u p e r v i s o r s , Dr. Juergen V i e l k i n d (Department o f Pathology and Environmental Carcinogenesis U n i t , B.C. Cancer Research Centre) and Dr. David Randall (Department o f Zoology), f o r t h e 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 ( F i s h e r i e s and Oceans, Canada), f o r t h e i r concern, encouragement, and help; to Dr. Bahram Sadaghiani f o r help w i t h embryo m i c r o i n j e c t i o n s ; and t o Ms. Barbara Schmidt, Mr. Bruce Woolcock, and Ms. Arlene S u l l i v a n , f o r expert t e c h n i c a l advice. I would s p e c i a l l y l i k e to thank the Canadian I n t e r n a t i o n a l Development Agency f o r p r o v i d i n g generous f i n a n c i a l support. This research was also supported by grants from the National I n s t i t u t e s o f Health (USA) and the Medical Research Council (Canada) to Juergen R. V i e l k i n d .  1 INTRODUCTION  The Xenopus oocyte and f e r t i l i z e d egg have been s u c c e s s f u l l y employed as t r a n s i e n t expression systems i n the a n a l y s i s of the temporal and s p a t i a l r e g u l a t i o n of gene expression during e a r l y 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 r e g u l a t i o n of Xenopus genes coding f o r 4S tRNA and 5S rRNA, and of sea u r c h i n 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 r e g u l a t i o n of a Xenopus g a s t r u l a - s p e c i f i c gene ( K r i e g and Melton, 1985) and the t i s s u e - s p e c i f i c r e g u l a t i o n of a Xenopus a c t i n gene (Wilson et  al., 1986).  Also commonly used i n t r a n s i e n t expression analyses i s the  f e r t i l i z e d sea u r c h i n egg, i n which ontogenic r e g u l a t i o n of sea u r c h i n genes coding f o r c y t o s k e l e t a l a c t i n (Davidson et al. , 1985; F l y t z a n i s et al. , 1987; Franks et al., 1988; Katula et al., 1987), and e a r l y and l a t e histones ( C o l i n 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 c y t o s k e l e t a l a c t i n gene was also observed i n t h i s system (Hough-Evans et al., 1987, 1988). Two major c h a r a c t e r i s t i c s of t r a n s i e n t expression systems, mosaicism and gradual l o s s of the introduced gene over time, l i m i t the scope of i n v e s t i g a t i o n s to which they can be a p p l i e d . Hough-Evans et al. (1988) point out, however, that mosaicism may  i n c e r t a i n circumstances be advantageous  since the presence of incorporated DNA only i n a percentage of c e l l s impinges l e s s 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 s t a b l y  r e t a i n at l e a s t one copy of the gene i n every c e l l are amenable, f o r example, to studies of c e l l lineage or phenotypic e f f e c t s of i n s e r t i o n a l mutagenesis, among others (reviewed i n Jaenisch, 1988).  I n t e g r a t i o n of introduced genes  and i n h e r i t a n c e by progeny, i . e . , the c r e a t i o n of s t a b l e transgenic l i n e s has been reported f o r several organisms, f o r example mice (reviewed i n Palmiter  2  and B r i n s t e r , 1986, and J a e n i s c h , 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 s t u d i e s . Transgenic technology has also been applied to the genetic manipulation of commercial l i v e s t o c k .  Cloned growth hormone genes have been introduced  i n t o c a t t l e (McEvoy et al., 1985; Nancarrow et al., f i s h (Zhu et al., Guyomard et al.,  1987; King and W a l l , 1988), sheep (Hammer et  1987), pigs and r a b b i t s (Hammer et al.,  1985, 1986; Chourrout et al., 1988).  al.,  1985), and  1986; Dunham et al., 1987;  The t r a n s f e r of a disease r e s i s t e n c e gene into  p o u l t r y (Crittenden and S a l t e r , 1985) and of a c o l d temperature tolerance gene i n t o f i s h (Hew et al.,  1987) have also been reported.  I n many instances,  s u c c e s s f u l i n t e g r a t i o n and or expression of the t r a n s f e r r e d genes has been demonstrated. The recent reports of gene t r a n s f e r i n t o f i s h (reviewed i n Maclean et al.,  1987) have been concerned w i t h improvement of c e r t a i n 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 t r a n s i e n t , and a l s o as a s t a b l e , expression system f o r t e s t i n g developmentally important f i s h genes, or any other genes with p o s s i b l e a p p l i c a t i o n s to the genetic engineering of commercial f i s h species.  The medaka appears to be an excellent  model f o r such studies since i t s b i o l o g y , i n c l u d i n g embryonic development and physiology, has been e x t e n s i v e l y studied, hundreds of eggs may be obtained d a i l y (Yamamoto, 1967, 1975), and a transparent chorion permits easy observation of embryonic development. attempts i n t o f i s h , Ozato et al.  I n one of the e a r l i e r gene t r a n s f e r  (1986) microinjected the chicken 5 - c r y s t a l l i n  gene i n t o 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 l o c a t i n g 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  a f t e r m i c r o i n j e c t i o n were r e q u i r e d .  In the present study, a s i m p l i f i e d  procedure of m i c r o i n j e c t i o n into the cytoplasm of f e r t i l i z e d medaka eggs p r i o r , to or immediately a f t e r f i r s t cleavage (one to two c e l l stage embryo) was adopted.  The chloramphenicol a c e t y l t r a n s f e r a s e (CAT) gene was chosen as a  reporter gene because simple and r a p i d t e s t s are a v a i l a b l e f o r t e s t i n g  CAT  a c t i v i t y , and because no s i m i l a r enzyme has been found i n eucaryotic systems. A double v i r a l promoter-enhancer c o n s i s t i n g of the Simian v i r u s 40 (SV 40) e a r l y region and the long terminal repeat (LTR) of the Rous sarcoma v i r u s (RSV) was chosen to regulate t r a n s c r i p t i o n 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 ( V i e l k i n d and Vogel, 1988). A number of reports have compared the expression of exogenous genes when a p p l i e d i n various conformations  i n vivo.  E t k i n and B a l c e l l s (1985) found  that s u p e r c o i l e d DNA  i n j e c t e d i n t o Xenopus embryos e x h i b i t e d higher expression  than d i d 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 t e s t whether DNA  topology has any e f f e c t 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 u n i t encompassed i n recombinant plasmid was i n j e c t e d i n e i t h e r s u p e r c o i l e d or l i n e a r and CAT expression monitored during medaka development.  conformation,  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 t r a n s f e c t i o n studies by I s h i u r a et al. (1982) and Okayama and Berg (1985) have shown that phage particle-mediated gene t r a n s f e r r e s u l t e d i n higher transformation rates i n mouse c e l l l i n e s than did DNA-mediated gene t r a n s f e r , 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 l a r g e r genes (15 to 20 kb) to be cloned, and phage are commonly used i n c l o n i n g genomic l i b r a r i e s .  Successful expression of the  DNA  c a r r i e d by the i n j e c t e d phage p a r t i c l e s not only would allow long stretches of genomic DNA  to be tested f o r the e f f e c t s of various introns and of d i s t a n t  4 regulatory regions on gene t r a n s c r i p t i o n and t r a 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 f o r expression i n medaka embryos since Wilson et al.  (1986) reported poor t r a n s c r i p t i o n f o r  recombinant phage DNA, p o s s i b l y due to i n t e r f e r e n c e by the arms of the phage vector. Several other studies have concentrated on the f a t e of exogenous DNA i n j e c t e d i n t o the f e r t i l i z e d egg, since c o r r e c t temporal or s p a t i a l expression depends, i n p a r t , on the s u r v i v a l and persistence of the DNA f o r a s u f f i c i e n t period of time f o r the gene to be regulated.  I t has been observed that l i n e a r  plasmid molecules, when i n j e c t e d i n t o the cytoplasm of f e r t i l i z e d Xenopus eggs, p e r s i s t f o r a longer period than do supercoiled molecules ( E t k i n et 1984; Wilson et al.,  1986; M a r i n i et al.,  1988).  al.,  In a d d i t i o n , Wilson et al.  (1986) observed that supercoiled DNA remained unchanged whereas l i n e a r plasmid DNA was processed i n t o high molecular weight concatenates.  M a r i n i 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 c o n s i s t i n g of h e a d - t o - t a i l tandem arrays and the l i n e a r plasmidderived species c o n s i s t i n g of e i t h e r h e a d - t o - t a i l or random a r r a y s . u r c h i n s , McMahon et al.  In sea  (1985) observed that only l i n e a r , but not supercoiled,  DNA was processed when i n j e c t e d i n t o the cytoplasm of f e r t i l i z e d eggs. et al.  Wilson  (1986) a l s o reported that a A clone c a r r y i n g a Xenopus cardiac a c t i n  gene p e r s i s t e d poorly i n the Xenopus embryo unless the i n s e r t was separated from the vector arms p r 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 e f f e c t on DNA v i a b i l i t y and processing i n the medaka embryo, supercoiled or l i 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 i n j e c t e d 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 t r a n s i e n t expression s t u d i e s , another major o b j e c t i v e i n gene t r a n s f e r experiments i s the production of stable transgenic animals, i . e . animals i n which i n j e c t e d exogenous DNA  i s incorporated i n t o the host genome  and i s transmitted through the germline to o f f s p r i n g . In mice, i t has been shown that only m i c r o i n j e c t i o n s i n t o the p r o n u c l e i of the f e r t i l i z e d r e s u l t e d i n high frequencies of DNA  egg  i n t e g r a t i o n , and nuclear i n j e c t i o n s  r e s u l t e d i n more e f f i c i e n t i n t e g r a t i o n when l i n e a r DNA molecules rather than s u p e r c o i l e d molecules were used (see B r i n s t e r et al., 1985).  Also, Costantini  and Lacy (1981) demonstrated that pronuclear m i c r o i n j e c t i o n of a A DNA  clone  r e s u l t e d i n high frequency of i n t e g r a t i o n i n t o mouse t i s s u e s and subsequent germline transmission.  In contrast to the almost absolute n e c e s s i t y f o r  nuclear i n j e c t i o n s i n mice, genomic i n t e g r a t i o n of a c y t o p l a s m i c a l l y i n j e c t e d l i n e a r plasmid DNA has been shown i n the sea u r c h i n ( F l y t z a n i s et al.,  1985),  and E t k i n and Pearman (1987) detected germline transmission of a supercoiled plasmid to o f f s p r i n g 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  i n j e c t e d into  the cytoplasm of f e r t i l i z e d t r o u t or z e b r a f i s h eggs p e r s i s t e d i n adult f i s h and was  i n h e r i t e d by a c e r t a i n percentage of o f f s p r i n g .  (1988) a l s o reported that supercoiled pUSVCAT DNA  V i e l k i n d et al.  cytoplasmically injected  i n t o f e r t i l i z e d z e b r a f i s h eggs r e s u l t e d i n stable transformants whose o f f s p r i n g not only i n h e r i t e d the f o r e i g n sequences but also expressed the gene. DNA,  CAT  To determine i f cytoplasmic i n j e c t i o n s of supercoiled or l i n e a r pUSVCAT  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, o f f s p r i n g from DNA  or phage-treated parents  were analysed by CAT enzyme assay f o r i n h e r i t e d expression of the CAT gene.  6  MATERIALS AND METHODS  Egg c o l l e c t i o n and embryo c u l t u r e C l u s t e r s of f e r t i l i z e d medaka eggs attached to the females were c o l l e c t e d 1 to 2 hr a f t e r the s t a r t of the l i g h t cycle and were t r a n s f e r r e d to and maintained i n Ringer's s o l u t i o n (0.75% NaCl, 0.02% KC1, 0.02% C a C ^ , pH 7.3; Yamamoto, 1961) f o r up to 2 hr a t 12 °C p r 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 c o n s i s t i n g of 1%  NaCl, 0.03% KC1, 0.04% CaCl 2 .2H 2 0, 0.163% MgS0^.7H20, 0.001% methylene blue (Kirchen and West, 1976). (1949).  Staging of medaka embryos was according to Matsui  Embryos that hatched were t r a n s f e r r e d 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 t h i s study.  The  t r a n s c r i p t i o n a l u n i t c o n s i s t s of the b a c t e r i a l CAT coding sequence under the r e g u l a t i o n of the Rous sarcoma v i r u s (RSV) LTR and Simian v i r u s 40 (SV 40) e a r l y region double promoter-enhancer regions.  This CAT t r a n s c r i p t i o n u n i t  was tested as a recombinant plasmid pUSVCAT (Karlsson et al., 1985) i n supercoiled form and also as a l i n e a r molecule a f t e r d i g e s t i o n with S a i l .  A  recombinant CAT phage p a r t i c l e containing three tandem copies of the CAT t r a n s c r i p t i o n u n i t was also used i n t h i s study ( V i e l k i n d and Vogel, 1988), as was the p u r i f i e d DNA from t h i s 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 e q u i l i b r i u m density c e n t r i f u g a t i o n s .  Recombinant CAT phage were  p u r i f i e d from p l a t e l y s a t e s by CsCl step gradient c e n t r i f u g a t i o n and were d i a l y s e d against SM b u f f e r (100 mM NaCl, 8 mM MgS0 4 .7H 2 0, 50 mM T r i s pH 7.5,  7  Figure 1  (a) Plasmid map of pUSVCAT showing CAT coding r e g i o n , promoterenhancer regions (*•-), and relevant r e s t r i c t i o n s i t e s . (b) pUSVCAT l i n e a r i z e d a t the S a i l s i t e . (c) Map of recombinant CAT phage DNA. The i n s e r t c o n s i s t s of three tandem copies of a 5200 bp BamHI fragment of pUSVCAT containing the CAT t r a n s c r i p t i o n r u n i t . A n , polyadenylation s i t e ; amp , a m p i c i l l i n r e s i s t a n c e ; 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 restriction site.  SV40ori  pUC9 An  RSV cat LTR  SV 40 ori K  EMBL3 arm  amp'' B  3 x BamHI fragments I I B B B CAT phage DNA (44.2 kb)  EM3L3 arm B  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 l i 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 microinjected i n t o 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.  I n j e c t i o n s were done under a b i n o c u l a r microscope (Zeiss)  w i t h a m a g n i f i c a t i o n range of 8x to 50x. Concentrations of 50 //g/ml plasmid DNA, 8 x 1 0 ^ phage p a r t i c l e s / m l (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 s o l u t i o n s to a f i n a l concentration of 0.25% to a i d i n estimation of i n j e c t i o n volume ( c a . 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).  I n d i v i d u a l embryos, h a t c h l i n g s , 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 T r i s pH 8.0, and then subjected to three 5 min cycles of freeze-thawing; extracts were obtained a f t e r c e n t r i f u g a t i o n (Eppendorf, 5 min, 4 °C) . To 100  Hi d H 2 ° .  2  e x t r a c t , 20  14  1  A*l C-chloramphenicol (NEN DuPont, 60 mCi/mmol, 100 / i d / m l ) , and  20 / i l 4 mM a c e t y l coenzyme A (Boehringer Mannheim) were added, and the mixture was incubated f o r 1 hr at 37 °C. The ^C-chloramphenicol and i t s acetylated forms were extracted w i t h 1 ml e t h y l a c e t a t e , d r i e d under vacuum, resuspended i n 30 / i l e t h y l a c e t a t e , and then spotted and separated on s i l i c a g e l chromatography p l a t e s (J.T. Baker Co.) f o r 50 min i n chloroform:methanol (95:5).  A f t e r the p l a t e s were a i r - d r i e d , autoradiograms were produced by  10  exposure o f X-ray f i l m (Kodak XAR-5) to these p l a t e s f o r one or seven days i n the presence o f an i n t e n s i f y i n g screen (DuPont).  DNA e x t r a c t i o n I n d i v i d u a l embryos, h a t c h l i n g s , and free-swimming f i s h , as w e l l as pools of ten embryos, were homogenized i n 200 pi o f l x SET (100 mM NaCl, 20 mM EDTA, 50 mM T r i s pH 7.8), 0.5% SDS, 0.5 mg/ml proteinase K and incubated f o r 2 to 4 hr a t 37 °C.  The samples were then extracted with one volume o f  phenol:chloroform:isoamyl a l c o h o l (25:24:1), r e - e x t r a c t e d w i t h an equal volume of butanol:isopropanol (7:3), p r e c i p i t a t e d with 2 volumes o f 95% ethanol f o r at l e a s t 2 h r at -20 °C and r e d i s s o l v e d overnight i n TE (10 mM T r i s pH 8.0, 1 mM EDTA).  Determination of embryo DNA content The genomic DNA content o f medaka embryos a t stages up to the e a r l y high b l a s t u l a stage was c a l c u l a t e d by m u l t i p l y i n g the amount o f 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 a t the embryonic stage.  For l a t e r developmental stages, DNA was  extracted from embryos and measured f l u o r o m e t r i c a l l y using the b i s benzimidazole (Hoechst 33258, Hoefer S c i e n t i f i c Instruments) method as s p e c i f i e d by the manufacturer.  Southern b l o t s T o t a l DNA from i n d i v i d u a l embryos, h a t c h l i n g s , or free-swimming f i s h , or a l i q u o t s o f DNA from pooled samples (equivalent to s i n g l e embryos o f the various s t a g e s ) , e i t h e r non-digested or completely digested by r e s t r i c t i o n enzymes as s p e c i f i e d by the manufacturer, were subjected to electrophoresis i n 0.8% agarose (Bio-Rad) g e l s .  Gels were soaked once i n 250 mM HC1 f o r 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 f o r 15 min to  11 denature DNA strands, and twice i n 1.5 M NaCl, 0.5 M T r i s pH 7.5 f o r 15 min to n e u t r a l i z e the pH of the g e l . A f t e r c a p i l l a r y t r a n s f e r of DNA onto nylon f i l t e r s (Schleicher & Schuell Nytran) i n 20x SSC b u f f e r (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 d r i e d in vacuo f o r 2 hr at 80 °C.  Hybridizations F i l t e r s were prehybridized f o r 15 min at 60 °C w i t h a s o l u t i o n containing 3x SSC, 10 mM T r i s pH 7.6, 10 mM EDTA, 0.5% SDS, l x Denhardt's (0.02% BSA, 0.02% p o l y v i n y l p y r o l l i d o n e , 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 h y b r i d i z e d overnight at 60 °C i n a s i m i l a r  s o l u t i o n c o n t a i n i n g pUSVCAT DNA, which had been l a b e l l e d by random hexamer priming (Feinberg and V o g e l s t e i n , 1983/84) w i t h a c t i v i t y of >5 x 10  J/:  P-dCTP to a s p e c i f i c  cpm//ig. The f i l t e r s were washed twice i n 2x SSC, 0.5%  SDS. f o r 30 min at room temperature (low s t r i n g e n c y ) , and twice i n O.lx SSC, 0.5% SDS f o r 30 min at 60°C (high s t r i n g e n c y ) .  Autoradiograms of the f i l t e r s  were then obtained by exposure of the d r i e d f i l t e r s to X-ray f i l m i n the presence of i n t e n s i f y i n g screens.  Quantitation of DNA h y b r i d i z a t i o n s i g n a l s H y b r i d i z a t i o n 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 c o n t a i n i n g 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 f o r 10 min each.  H y b r i d i z a t i o n bands c o n t a i n i n g known  amounts of DNA were a l s o counted i n the same manner and used as a reference to convert cpm (counts per minute) values to picogram DNA amounts.  12  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 i n d i v i d u a l s or pooled batches at various stages from the e a r l y 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 r e s u l t s of the CAT enzyme assay of i n d i v i d u a l medaka of various  stages derived from f e r t i l i z e d eggs i n j e c t e d with supercoiled pUSVCAT DNA i s shown i n Figure 2a.  A l t o g e t h e r , ten i n d i v i d u a l s at each stage of development  were assayed, of which representative r e s u l t s are shown.  CAT enzyme a c t i v i t y  was not d e t e c t i b l e i n the 32-64 c e l l stage embryo (lane 1, 2 hr postinjection).  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 o l d 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 v a r i e d n o t i c e a b l y among the h a t c h l i n g s assayed (lane 810, 2 weeks p o s t - i n j e c t i o n ) . was  By the free-swimming f i s h stage, CAT  activity  s t i l l d e t e c t i b l e i n a few of the f i s h assayed, one of which had a  moderately strong s i g n a l (lane 11-13, 4 weeks p o 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 t r e a t e d embryos i s not due to expression of endogenous genes.  S l e i g h (1986)  and Crabb and Dixon (1987) have reported the presence of substances i n c e l l e x t r a c t s that i n t e r f e r e 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 d i f f e r e n t stages to which equal amounts of commercially a v a i l a b l e CAT enzyme had. been added.  No  13  Figure 2  CAT gene expression a f t e r i n j e c t i o n of (a) s u p e r c o i l e d , and (b) l i n e a r , pUSVCAT DNA i n t o f e r t i l i z e d medaka eggs. Eggs were i n j e c t e d with 25 pg of e i t h e r plasmid form and allowed to develop u n t i l harvested. I n d i v i d u a l s at various developmental stages were harvested and assayed f o r 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, d i a c e t y l a t e d CM.  Stages:  32-64  late g a s t r u l a /  cells  early neurula  one-week embryo  old  hatchling  free-swimming fish  15  d e t e c t i b l e d i f f e r e n c e s i n CAT a c t i v i t y s i g n a l were observed among these c o n t r o l groups (data not shown), i n d i c a t i n g that the observed p a t t e r n of CAT s i g n a l s obtained by i n j e c t i n g supercoiled pUSVCAT DNA i s not a r e s u l t of d i f f e r e n t i n h i b i t o r y c a p a c i t i e s of the embryonic stages, but represents the CAT expression p a t t e r n of the introduced gene. For embryos i n j e c t e d w i t h S a i l - l i n e a r i z e d pUSVCAT ( F i g 2b), an expression p a t t e r n s i m i l a r to that f o r supercoiled pUSVCAT i n j e c t e d embryos was observed, but w i t h marginally stronger s i g n a l s at the gastrula/neurula stage and s l i g h t l y weaker s i g n a l s at the h a t c h l i n g 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 r e s u l t s of the CAT expression experiments using CAT phage p a r t i c l e s  i s shown i n Figure 3a. No CAT expression was d e t e c t i b l e i n embryos assayed at the 32-64 c e l l stage (lane 1 ) , but weak CAT expression was observed i n several l a t e g a s t r u l a / e a r l y neurula stage embryos (lane 2-4).  A s l i g h t l y stronger CAT  expression was c o n s i s t e n t l y observed i n one-week o l d embryos (lane 5-7), and CAT expression was e i t h e r weak or not detected at the h a t c h l i n g stage (lane 810).  However, weak CAT expression was s t i l l d e t e c t i b l e i n a few free-swimming  stage f i s h (lane 11-13). I n j e c t i o n s w i t h p u r i f i e d CAT phage DNA r e s u l t e d i n a p a t t e r n of CAT gene expression s i m i l a r to that obtained w i t h CAT phage p a r t i c l e s ( F i g 3b). However, g e n e r a l l y stronger s i g n a l s were observed, presumably r e s u l t i n g from the higher CAT phage DNA concentration used, which was the equivalent of f i v e 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 c o n s i s t e n t l y detected i n the l a t e g a s t r u l a / e a r l y neurula (lane 2-4), w h i l s t one-week o l d 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 s i g n a l s were generally  16  Figure 3  CAT gene expression a f t e r 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, i n t o f e r t i l i z e d medaka eggs. Eggs were i n j e c t e d 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. I n d i v i d u a l s at various developmental stages were harvested and assayed f o r 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  11  10  12  13  Ac -CM 3  Ac CM r  • • t f f ©  b  1  ©  ©  2  3  ©  4  • t f t 6  I Stages:  •  •  •  ©  ©  5  6  7  ©  8  ••• ©  ©  ©  I  •  9  early neurula  one-week embryo  old  •  10  11  •  12  •  13  • • • • • • ©  ©  ©  +  32-64 l a t e g a s t r u l a / cells  ©  »  •  + hatchling  free-swimming fish  CM  18 weaker and v a r i e d widely i n i n d i v i d u a l h a t c h l i n g s (lane 8-10), and were d e t e c t i b l e only i n a few of the free-swimming f i s h t e s t e d (lane 11-13).  1.3  Onset of CAT gene expression i n the e a r l y medaka embryo The very strong CAT expression observed i n gastrula/neurula stage  embryos derived from treatment w i t h e i t h e r supercoiled or l i n e a r pUSVCAT DNA suggest that CAT expression began a t 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 l a t e g a s t r u l a / e a r l y neurula stage on pooled batches of 3 embryos ( F i g 4 ) . Expression appeared to begin from the e a r l y high b l a s t u l a stage (lanes 2, 4 hr p o s t - i n j e c t i o n ) , a c l e a r s i g n a l being apparent by the f l a t b l a s t u l a stage (lanes 3, 10 h r p o s 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 p o s t - i n j e c t i o n ) .  No d i f f e r e n c e s were  observed between the supercoiled and l i 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 b l o t a n a l y s i s of DNA samples obtained from i n d i v i d u a l s or pools a t 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 i n j e c t e d supercoiled and l i n e a r plasmid DNA The r e s u l t s of the Southern b l o t analyses of i n d i v i d u a l medaka DNA,  a r 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 s u p e r c o i l s , 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 l a t e g a s t r u l a / e a r l y  neurula stage an a d d 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 a f t e r i n j e c t i o n of (a) s u p e r c o i l e d , and (b) l i n e a r , pUSVCAT DNA. 25 pg of e i t h e r plasmid form was i n j e c t e d i n t o each f e r t i l i z e d egg. At various e a r l y embryonic stages, 3 samples were pooled together and assayed f o r the presence of CAT enzyme a c t i v i t y .  20  gastrula/neurula  *  •  •  •  flat  blastula  e a r l y high b l a s t u l a  32-64  cells  a  gastrula/neurula  flat  blastula  e a r l y high b l a s t u l a  32-64  m CD  o  cells  21  Figure 5  Southern b l o t a n a l y s i s of the fate of i n j e c t e d (a) s u p e r c o i l e d , 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 i n j e c t e d w i t h 25 pg of e i t h e r plasmid form. DNA was extracted from i n d i v i d u a l s at various developmental stages, subjected to e l e c t r o p h o r e s i s i n 0.8% agarose g e l s , and t r a n s f e r r e d to nylon membranes. The b l o t s were probed then with pUSVCAT DNA l a b 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). s c , 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; I n , l i n e a r pUSVCAT DNA; hmw, high molecular weight pUSVCAT DNA. Lambda H i n d l l l s i z e standards (kb) are shown at r i g h t of panel ( a ) .  22 a  1  b  1  2  3  2  4  3  5  4  5  6  6  7  7  10 11  8  9  8  9 10  12 13  11 12 13  C  In  I  Stages:  1  1  I  32-  'ate  one-week  64  gastruia/  old embryo  cells  early neurula  1  hatchling  1  freeswimming fish  23  kb was c l e a r l y evident (lane 2-4), co-migrating w i t h the high molecular weight f r a c t i o n of medaka DNA (as seen by ethidium bromide s t a i n i n g of the gel p r i o r 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, w i t h the high molecular weight form showing the greatest increase. Strong h y b r i d i z a t i o n smears were also observed, suggesting degradation of a s i g n i f i c a n t percentage of plasmid DNA.  I n the one-week o l d embryo, the open  c i r c u l a r and multimeric forms could not be detected, some s u p e r c o i l e d plasmid was s t i l l evident, and the r e s t of the plasmid DNA was of the high molecular weight form (lane 5-7); plasmid DNA degradation was s t i l l evident. I n h a t c h l i n g s , plasmid DNA was f u r t h e r reduced and only the h i g h molecular weight form remained (lane 8-10).  Input plasmid DNA p e r s i s t e d i n one of s e v e r a l  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 s t r o n g l y h y b r i d i z i n g high molecular weight form ( F i g 5b, lane 1 ) , suggesting a f a i r l y r a p i d conversion of the i n j e c t e d l i n e a r molecules. begun.  A f a i n t smear suggests that plasmid DNA degradation had already  I n the l a t e g a s t r u l a / e a r l y neurula, a very strong h y b r i d i z a t i o n i n the  high molecular weight band was observed, i n d i c a t i n g a large increase i n plasmid DNA.  A strong h y b r i d i z a t i o n smear was also apparent, suggesting  s u b s t a n t i a l degradation of the plasmid DNA.  Only the high molecular weight  form was present i n the one-week o l d embryo, and plasmid degradation was s t i l l evident (lane 5-7).  I n h a t c h l i n g s , the amount of plasmid DNA present was  f u r t h e r 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 h y b r i d i z a t i o n s i g n a l s already seen a t the gastrula/neurula stage i n both the s u p e r c o i l e d and l i n e a r pUSVCAT experiments suggest that processing of the i n j e c t e d plasmids must have begun e a r l i e r . order to have an idea of the r a p i d i t y of plasmid conversion, embryos at  In  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 a t each stage were pooled, and Southern b l o t a n a l y s i s  was performed on embryo-equivalent DNA a l i q u o t s a f t e r complete d i g e s t i o n with X h o l , which does not recognize any s i t e on pUSVCAT DNA.  D i g e s t i o n with Xhol  reduces high molecular weight medaka DNA t o smaller fragments, thus minimizing any p o s s i b l e impedance of pUSVCAT DNA migration during e l e c t r o p h o r e s i s .  DNA  extracted from 1-2 c e l l stage embryos w i t h i n 5 min of i n j e c t i o n w i t h supercoiled pUSVCAT DNA contained the expected s u p e r c o i l e d , open c i r c u l a r , and multimeric c i r c u l a r forms ( F i g 6a, lane 1 ) .  The high molecular weight form,  which was not d i f f e r e n t from that observed before, became apparent at the e a r l y high b l a s t u l a stage, and an o v e r a l l increase i n the other pUSVCAT forms was seen (lane 2, 4 h r p o s t - 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 p o s t - i n j e c t i o n ) , the high molecular weight form became more prominent than the other forms, suggesting p r e f e r r e d r e p l i c a t i o n of the high molecular weight form.  I n a d d i t i o n , a strong smear was observed, s i g n a l l i n g the onset  of plasmid DNA degradation.  The strongest o v e r a l l DNA h y b r i d i z a t i o n s i g n a l  was observed i n the l a t e g a s t r u l a / e a r l y neurula, as p r e v i o u s l y noted (lane 4 , 1 day p o s t - i n j e c t i o n ) 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 d e c l i n e d (lane 5, 3 days p o s t - i n j e c t i o n ) . In c o n t r a s t to the experiment with supercoiled pUSVCAT DNA, much of the l i n e a r pUSVCAT DNA i n j e c t e d i n t o f e r t i l i z e d eggs was almost immediately converted to high molecular weight form ( F i g 6b, lane 1, 5 min postinjection).  I n a d d i t i o n , f a i n t bands corresponding to s u p e r c o i l e d and open  c i r c u l a r pUSVCAT forms could be made out, i n d i c a t i n g some conversion of the l i n e a r DNA to these forms too.  I n the e a r l y high b l a s t u l a stage, the amounts  of s u p e r c o i l e d , open c i r c u l a r , and e s p e c i a l l y high molecular weight pUSVCAT forms increased while that of the l i 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 f u r t h e r increased.  The strongest DNA  25  Figure 6  Southern b l o t a n a l y s i s of the e a r l y fate of i n j e c t e d (a) supercoiled, and (b) l i n e a r , pUSVCAT molecules. F e r t i l i z e d eggs i n j e c t e d with e i t h e r supercoiled or l i n e a r pUSVCAT DNA were harvested a t various e a r l y embryonic stages, and t o t a l DNA was obtained from pools of ten embryos. Embryo-equivalent a l i q u o t s were subjected to e l e c t r o p h o r e s i s , b l o t t e d , h y b r i d i z e d to radiol a b e l l e d pUSVCAT probe, and autoradiographed as described i n F i g 5. s t d , 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  CM  O  (fl  30 somites  in  CO  I I  1 1 t  i-  gastrula/neurula flat blastula early high blastula 1-2  cells  30 somites  * I  gastrula/neurula  CO  flat blastula  CM  early high blastula 1-2  cells  03  W 0) CO CO  OT  27  h y b r i d i z a t i o n s i g n a l and smearing was seen i n the l a t e g a s t r u l a / e a r l y 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 f a c t that the migration of the high molecular weight plasmid form  was unaffected by d i g e s t i o n of the embryo DNA samples w i t h X h o l , which reduces genomic DNA to smaller fragment s i z e s 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 s u p e r c o i l e d , 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 t h i s high molecular weight pUSVCAT form, Southern b l o t analysis was performed on DNA from h a t c h l i n g s , a stage i n which the remaining pUSVCAT DNA had e a r l i e r been observed to be e x c l u s i v e l y of the high molecular weight form.  DNA from i n d i v i d u a l hatchlings was digested w i t h Kpnl and analysed by  Southern b l o t h y b r i d i z a t i o n using r a d i o a c t i v e l y 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. I n h a t c h l i n g s derived from eggs i n j e c t e d with supercoiled pUSVCAT DNA, d i g e s t i o n of the DNA produced a s i n g l e h y b r i d i z a t i o n band corresponding to a 7.9 kb l i n e a r monomer ( F i g . 7a). This r e s u l t suggests that the high molecular weight plasmid form derived from supercoiled plasmid i n j e c t i o n s probably consisted of tandem arrays of plasmid monomers oriented h e a d - t o - t a i l .  However i n  h a t c h l i n g s derived from eggs i n j e c t e d with l i n e a r pUSVCAT DNA, three strong h y b r i d i z a t i o n bands of approximately 9.8 kb, 7.9 kb, and 6.0 kb were seen ( F i g . 7b,c). I n a d d i t i o n , two other very weak h y b r i d i z a t i o n 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 i n d i c a t e s that the resident high molecular weight plasmid form c o n s i s t e d of randomly oriented tandem arrays of the l i n e a r plasmids.  Kpnl  d i g e s t i o n of such a tandem array would be expected to y i e l d many copies of  28  Figure 7  Southern b l o t a n a l y s i s of high molecular weight pUSVCAT DNA p e r s i s t i n g i n h a t c h l i n g s . Hatchlings derived from eggs i n j e c t e d with ( a ) s u p e r c o i l e d , or (b) l i n e a r , pUSVCAT DNA were i n d i v i d u a l l y harvested f o r t h e i r DNA. The DNA samples were digested with Kpnl, subjected to e l e c t r o p h o r e s i s , b l o t t e d , h y b r i d i z e d to radiol a b e l l e d pUSVCAT probe, and autoradiographed as described i n F i g 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 r e s u l t i n g from Kpnl d i g e s t i o n of S a i l l i n e a r i z e d pUSVCAT. (d) Diagram of a h y p o t h e t i c a l multimer of S a i l l i n e a r i z e d pUSVCAT DNA showing a l l p o s s i b l e ( h e a d - t o - t a i l , 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 j u n c t i o n s . The predicted s i z e (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) d i g e s t i o n are i n d i c a t e d .  29  O) ^  0)  o CO  T3 tn  CO  I  CN  I  ai  OQ  if)  CO  CM  CO 05 O 05 0>N" CO «tf  o pj  9  in  CO CM  O)  *  30  middle fragments w i t h s i z e s represented by the three strong h y b r i d i z a t i o n bands, and fewer copies of end fragments corresponding to the weak h y b r i d i z a t i o n bands ( F i g 7d).  The r e l a t i v e l y stronger h y b r i d i z i n g s i g n a l of  the u n i t length band compared to the other two middle fragment bands implies that a m a j o r i t y of the l i g a t i o n j u n c t i o n s were of the h e a d - t o - t a i l type.  2.2  Conformational changes of DNA introduced w i t h i n recombinant phage p a r t i c l e s and of p u r i f i e d phage DNA The r e s u l t s of the Southern b l o t analyses of i n d 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 a l s o present i n the l a t e g a s t r u l a / e a r l y 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 h y b r i d i z a t i o n s i g n a l w i t h some very l i g h t smearing, i n d i c a t i v e of concurrent f o r e i g n DNA r e p l i c a t i o n and degradation.  This observation therefore suggests  that DNA contained w i t h i n the i n j e c t e d 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 o l d embryo, the CAT phage DNA appeared to co-  migrate w i t h the high molecular weight f r a c t i o n of medaka DNA (lane 5-7). O v e r a l l h y b r i d i z a t i o n s i g n a l was only s l i g h t l y weaker, but smears were more visible.  CAT phage DNA was f u r t h e r reduced i n h a t c h l i n g s (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 i n j e c t e d with p u r i f i e d CAT phage DNA and analysed at the 32-64 c e l l stage a l s o showed a band corresponding to u n i t length CAT phage DNA ( F i g 8b, lane 1 ) .  By the gastrula/neurula stage, a stronger h y b r i d i z a t i o n band was  observed, i n d i c 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 a l s o occurred.  In the  one-week o l d embryo, there seems to have been a s h i f t toward higher molecular weight i n the DNA h y b r i d i z a t i o n 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 b l o t analysis of the fate of (a) DNA o f i n j e c t e d CAT phage p a r t i c l e s and (b) i n j e c t e d CAT phage DNA molecules during medaka development. F e r t i l i z e d eggs were i n j e c t e d w i t h 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 i n d i v i d u a l s at various developmental stages, subjected to e l e c t r o p h o r e s i s , b l o t t e d , h y b r i d i z e d to r a d i o - l a b e l l e d pUSVCAT probe, and autoradiographed as described i n F i g 5. Arrows p o i n t to CAT phage DNA monomer. Lambda H i n d l l l s i z e standards (kb) are shown at r i g h t 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  -«  b  1  2  3  4  5  4*  I Stages:  1  3 2  ~  64 cells  6  7  gastrula/ early neurula  9  10  11 12 13  •  1 late  8  1 one-week old embryo  hatchling  1  1 freeswimming fish  2.3 2.0  33  s i g n i f i c a n t l y reduced and no longer d e t e c t i b l e i n several i n d i v i d u a l s at the h a t c h l i n g stage (lane 8-10), but continued to p e r s i s t to the free-swimming stage i n a t l e a s t one of the f i s h tested (lane 11-13).  2.3  R e p l i c a t i o n of i n j e c t e d f o r e i g n DNA sequences i n e a r l y embryos As already observed i n the Southern analyses of DNA from embryos  i n j e c t e d w i t h supercoiled and l i 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 t h e r e a f t e r .  I n the  experiments using supercoiled or l i n e a r plasmid DNA, the increasing i n t e n s i t i e s of DNA h y b r i d i z a t i o n s i g n a l s from the 1-2 c e l l stage to the gastrula/neurula stage suggest that the i n j e c t e d sequences were r e p l i c a t e d during the cleavage and g a s t r u l a t i o n stages of medaka embryogenesis.  At the  same time, the h y b r i d i z a t i o n i n t e n s i t y of the smears i n d i c a t e that a f r a c t i o n of the h y b r i d i z a t i o n s i g n a l consisted of degraded f o r e i g n DNA. In an attempt to determine the degree of f o r e i g n DNA r e p l i c a t i o n during e a r l y embryogenesis, a Southern b l o t h y b r i d i z a t i o n 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 i n j e c t e d 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 f u n c t i o n a l ) pUSVCAT monomers, whereas f o r eggs i n j e c t e d w i t h CAT phage DNA, a S a i l band migrating as a 15.6 kb molecule corresponds to i n t a c t CAT i n s e r t ( i . e . 3 tandemly arranged CAT t r a n s c r i p t i o n units). In experiments using supercoiled pUSVCAT DNA, t o t a l pUSVCAT DNA increased approximately t e n - f o l d by the l a t e g a s t r u l a / e a r l y neurula stage (22 hr p o s t - i n j e c t i o n ) , but the amount of i n t a c t pUSVCAT molecules a t t h i s stage represented only a s i x - f o l d increase ( F i g 9b & d ) .  Of the t o t a l pUSVCAT  sequences present at t h i s stage, 50% were recovered as u n i t length molecules.  34  Figure 9  Rapid increase and subsequent decrease of input pUSVCAT DNA during e a r l y embryogenesis, as measured by Southern b l o t h y b r i d i z a t i o n assay. Embryo-equivalent a l i q u o t s of the DNA samples generated i n the experiments i n F i g 6 were digested w i t h S a i l , subjected to e l e c t r o p h o r e s i s , b l o t t e d , h y b r i d i z e d to r a d i o - l a b e l l e d pUSVCAT probe, and autoradiographed as described i n F i g 5 . H y b r i d i z a t i o n bands on each f i l t e r lane corresponding to u n i t i n t a c t l i n e a r pUSVCAT molecules were cut out and q u a n t i f i e d 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 c a l c u l a t e d from the counts obtained from the e n t i r e lane. (a) and (b) T o t a l pUSVCAT DNA per embryo a f t e r i n j e c t i o n of f e r t i l i z e d eggs with l i n e a r and supercoiled pUSVCAT DNA, r e s p e c t i v e l y . (c) and (d) i n t a c t pUSVCAT DNA per embryo a f t e r i n j e c t i o n w i t h l i n e a r and supercoiled pUSVCAT DNA, r e s p e c t i v e l y . The time of harvest, embryonic stage, and genomic DNA content of each stage are i n d i c a t e d on the a b s c i s s a .  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 i n t a c t molecules were approximately n i n e - f o l d greater than that a t the time of i n j e c t i o n ( F i g 9a & c ) .  I n t a c t pUSVCAT u n i t s also c o n s t i t u t e d 50% of the  t o t a l pUSVCAT DNA sequences present a t 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 t h r e e - f o l d , but i n t a c t CAT i n s e r t increased d).  l e s s than one-fold, by the f l a t b l a s t u l a stage ( F i g 10b &  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; i n t a c t CAT i n s e r t  increased only t h r e e - f o l d and accounted f o r roughly 30% of the t o t a l CAT DNA sequences a t the gastrula/neurula stage ( F i g 10a & c ) . By the 30 somite stage of embryonic development (72 h r p o s t - i n j e c t i o n ) , 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 i n t a c t CAT DNA.  These r e s u l t s r e f l e c t a change i n  r e p l i c a t i o n of the f o r e i g n sequences r e l a t i v e to t h 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 a f t e r 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 a t diminishing rates of increase w i t h progressing development.  3.  I n h e r i t e d expression of CAT reporter gene i n o f f s p r i n g One of the o b j e c t i v e s i n t h i s study was to determine i f the i n j e c t e d DNA  or phage would r e s u l t i n genomic i n t e g r a t i o n of the f o r e i g n DNA as w e l l as i n the i n h e r i t a n c e and expression of these sequences by progeny f i s h .  I n order  to i d e n t i f y p o s i t i v e founder f i s h , several treated f i s h were out-crossed with untreated medaka. One-week o l d F-^ embryos or F-^ h a t c h l i n g s obtained from each out-cross were harvested and pooled i n batches of f i v e and analysed f o r  37  Figure 10  Increase and subsequent decrease of input CAT phage DNA during e a r l y embryogenesis. F e r t i l i z e d eggs were i n j e c t e d 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. T o t a l DNA was extracted from pools of ten embryos at various e a r l y embryonic stages. Embryoequivalent a l i q u o t s were digested w i t h S a i l , subjected to e l e c t r o p h o r e s i s , b l o t t e d , h y b r i d i z e d to r a d i o - l a b e l l e d pUSVCAT probe and autoradiographed as described i n F i g 5. H y b r i d i z a t i o n bands i n each f i l t e r lane corresponding to i n t a c t CAT DNA i n s e r t s , and the remainder of each lane (smears), were separately q u a n t i f i e d by s c i n t i l l a t i o n counting as described i n F i g 9. Total CAT DNA present i n each embryo was c a l c u l a t e d on a s i m i l a r basis as i n F i g 9. (a) and (b) T o t a l CAT DNA per embryo a f t e r i n j e c t i o n w i t h p u r i f i e d CAT phage DNA and recombinant CAT phage p a r t i c l e s , r e s p e c t i v e l y . (c) and (d) i n t a c t CAT DNA i n s e r t s per embryo a f t e r 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.  38  39 i n h e r i t e d CAT gene expression.  Representative CAT assays of pooled F-^ batches  from s e v e r a l parents derived from eggs c y t o p l a s m i c a l l y i n j e c t e d with s u p e r c o i l e d and l i 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 o f f s p r i n g r e s p e c t i v e l y , were obtained from each outc r o s s , and analysed f o r CAT expression, the r e s u l t s of which are l i s t e d i n Tables 1 through 4. CAT-positive o f f s p r i n g from parents representing a l l four i n j e c t i o n groups (supercoiled pUSVCAT, l i n e a r pUSVCAT, recombinant CAT phage, and CAT phage DNA) were detected.  CAT expression s i g n a l s v a r i e d 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 p o s i t i v e while other batches from the same parent were negative i n d i c a t e that the percentage of CAT-positive  offspring  was low, and that the germline-positive parents were probably mosaic f o r the CAT sequences.  40  Figure 1 1  I n h e r i t e d CAT gene expression i n pooled o f f s p r i n g of pUSVCAT DNAtreated parents. F e r t i l i z e d eggs that had been i n j e c t e d with (a) s u p e r c o i l e d , and (b) l i n e a r , pUSVCAT DNA were reared to maturity and outcrossed with untreated f i s h . O f f s p r i n g from each outcross were pooled i n t o batches of 5 and assayed f o r the presence of CAT enzyme a c t i v i t y . Autoradiograms show representative CAT assays of pooled F^ from 5 d i f f e r e n t parents f o r each treatment group. Alpha-numerals at the bottom of each lane i d e n t i f y the d i f f e r e n t parents. See Table 1 and 2 f o r d e t a i l e d r e s u l t s of F-^ CAT assays.  41  42  Figure 1 2  I n h e r i t e d CAT gene expression i n pooled o f f s p r i n g of CAT phage p a r t i c l e and CAT phage DNA t r e a t e d parents. F e r t i l i z e d eggs that had been i n j e c t e d w i t h (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 w i t h untreated f i s h . O f f s p r i n g were pooled and assayed f o r CAT a c t i v i t y , and autoradiograms of representative CAT assays shown, as described i n F i g 1 1 . See Table 3 and 4 f o r d e t a i l e d r e s u l t s of F-L CAT assays.  44  Table 1. I n h e r i t e d expression of supercoiled pUSVCAT DNA i n o f f s p r i n g . Medaka eggs i n j e c t e d with supercoiled pUSVCAT DNA were reared to adulthood and outcrossed w i t h untreated f i s h . O f f s p r i n g were pooled into batches of 5 and assayed f o r CAT enzyme a c t i v i t y , m, male; f , female; -, no d e t e c t i b l e 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  o  Sex  SI  m  +  +  nd  S2  m  -  -  -  S3  m  -  -  -  S4  m  -  +  -  S5  f  +  +  -  S6  f  +  +  +  S7  f  +  +  -  S8  f  -  +++  +  S9  f  +  +  -  S10  f  +  +  +  Sll  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  ++  ++  ++  F  45  Table 2. I n h e r i t e d expression of l i n e a r pUSVCAT DNA i n o f f s p r i n g . Parents were derived from eggs i n j e c t e d w i t h l i n e a r pUSVCAT DNA. Procedure f o r screening of o f f s p r i n g as described i n Table 1. F-^ batch no. *0-  Sex  Ll  m  nd  L2  m  nd  L3  m  L4  m  L5  f  L6  f  +++  L7  f  +  L8  m  +  +++  +  nd  L9  46 Table 3. I n h e r i t e d CAT expression i n o f f s p r i n g of parents derived from f e r t i l i z e d eggs i n j e c t e d w i t h recombinant CAT phage p a r t i c l e s . Procedure f o r screening of o f f s p r i n g as described i n Table 1. F-^ batch no. FQ  Sex  PI  f  P2  f  P3  m  P4  m  P5  -  -  nd  +  +++  m  -  +  P6  f  +++  +  P7  m  -  -  +  P8  f  ++  ++  ++  P9  m  -  -  -  P10  f  -  -  +  Pll  f  +  ++  +  47  Table 4. Inherited CAT expression i n o f f s p r i n g of parents derived from f e r t i l i z e d eggs i n j e c t e d with p u r i f i e d CAT phage DNA. Procedure f o r screening of o f f s p r i n g as described i n Table 1.  F  o  1  F-^ batch no. 2  3  4  Sex  Dl  f  +  +  +  +  D2  f  ++  ++  +  +  D3  f  +  ++  -  -  D4  f  +  +  +  +  D5  f  ++  ++  ++  nd  48 DISCUSSION  The expression and fate of a CAT reporter gene a f t e r m i c r o i n j e c t i o n 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 a p p l i e d as supercoiled or l i 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 l i n e a r pUSVCAT DNA r e s u l t e d i n peak CAT gene expression at the gastrula/neurula stage (1 day p o 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 o l d embryo. Expression was s i g n i f i c a n t l y reduced, and v a r i e d n o t i c e a b l y , i n the hatchlings sampled (2 weeks p o s t - i n j e c t i o n ) .  By the free-swimming f i s h stage (4 weeks  p o 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 w i t h s u p e r c o i l e d and l i n e a r plasmid DNA i n d i c a t e that p h y s i c a l conformation of input DNA has no s i g n i f i c a n t e f f e c t on i t s a b i l i t y to be expressed.  These r e s u l t s contrast  w i t h two c o n f l i c t i n g observations i n the Xenopus embryo; E t k i n and B a l c e l l s (1985) reported a higher expression f o r 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 a c t i n - g l o b i n f u s i o n gene were e f f i c i e n t l y t r a n s c r i b e d only i f the c i r c u l a r plasmid containing e i t h e r gene was f i r s t l i n e a r i z e d . The CAT expression p a t t e r n obtained when medaka eggs were i n j e c t e d 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 w i t h pUSVCAT DNA; peak CAT gene expression d i d not occur u n t i l the one-week o l d embryonic stage. Expression c h a r a c t e r i s t i c s at the h a t c h l i n g and free-swimming f i s h stages, however, were s i m i l a r to that observed w i t h 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 o l d embryo stage i s s u r p r i s i n g 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 t h e r e a f t e r . With CAT phage p a r t i c l e s , a s i m i l a r p a t t e r n of i n c r e a s i n g CAT a c t i v i t y up to the oneweek o l d embryo stage and subsequent decline was observed.  These observations  suggest a p o s s i b l e r o l e of the phage vector arms i n post-gastrula/neurula enhancement of gene expression.  In an experiment i n v o l v i n g the m i c r o i n j e c t i o n  of a lambda DNA clone i n t o f e r t i l i z e d Xenopus eggs, c o r r e c t l y 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 i n s e r t and the vector arms was i n j e c t e d , but not when the i n t a c t 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  e f f e c t on t r a n s c r i p t i o n of the a c t i n gene i n the Xenopus embryo. A n a l y s i s of embryos at e a r l i e r stages revealed that the i n j e c t e d s u p e r c o i l e d or l i n e a r pUSVCAT DNA was not expressed p r i o r to the e a r l y high b l a s t u l a stage (4 h r p o s t - i n j e c t i o n ) although approximately corresponding  25 pg DNA,  to 3 x 10^ copies of the CAT gene, was i n j e c t e d i n t o each 1-2  c e l l stage embryo.  The onset of CAT expression i n medaka embryos only a f t e r  the e a r l y 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 i n j e c t e d 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 i n j e c t e d 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. E t k i n and B a l c e l l s (1985), using the pSV2CAT plasmid DNA, were a l s o unable to detect expression of the CAT gene p r 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 midb l a s t u l a t r a n s i t i o n 8 hr a f t e r i n j e c t i o n was simply a consequence of increase i n pSV2CAT DNA copy number a f t e r 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 i n t o n o n - r e p l i c a t i n g oocytes e l i c i t e d expression as e a r l y as 2.5 hr a f t e r i n j e c t i o n .  CAT  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 w i t h a t r a n s - a c t i v a t i n g enhancing f a c t o r d i d not induce e a r l i e r expression, but d i d induce a much stronger expression at the mid-blastula t r a n s i t i o n . higher DNA  In the present study, i n j e c t i o n of  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 ( V i e l k i n d et al. , 1988). phage DNA  Although CAT  i n j e c t e d eggs were not analysed at the stages e a r l i e r than the  gastrula/neurula stage, the moderately strong s i g n a l seen at t h i s stage implies that the expression of the input CAT phage DNA embryonic stage.  a l s o began an e a r l i e r  In marked c o n t r a s t , 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 s i m i l a r  to the Xenopus mid-blastula t r a n s i t i o n i s not present during sea u r c h i n embryogenesis .  For example, sea u r c h i n e a r l y histone genes are expressed  during e a r l y 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 f o r a few cleavages  (Poccia et a l . , 1981).  al.,1988) and e a r l y histone H2B  I n j e c t e d e a r l y histone H2A ( C o l i n et al., 1988)  ( V i t e l l i et  genes showed peak  t r a n s c r i p t i o n together with endogenous genes during the e a r l y b l a s t u l a stage. As noted f o r a l l four treatment groups, f o r each of the stages up to the one-week o l d embryo stage, CAT expression s i g n a l s were h i g h l y consistent among the samples analysed.  At the h a t c h l i n g stage, s i g n a l strengths among the  samples v a r i e d n o t i c e a b l y , 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 r e f l e c t e d i n the f a t e of the introduced plasmid and phage DNA sequences whereby, at embryonic stages up to the one-week o l d embryo stage, the amount of f o r e i g n DNA  sequences present at each stage was  consistent among the sampled embryos. Amounts of exogenous DNA  fairly increased  during cleavage and g a s t r u l a t i o n and were s i g n i f i c a n t l y reduced by the one-  51 week o l d embryo stage.  Thereafter, DNA amounts f l u c t u a t e d among the sampled  h a t c h l i n g s , and was no longer d e t e c t i b l e i n most of the sampled free-swimming fish.  Upon i n j e c t i o n , supercoiled pUSVCAT DNA was gradually converted to a  high molecular weight concatemer oriented h e a d - t o - t a i l .  This high molecular  weight form was observed to increase g r e a t l y during cleavage and g a s t r u l a t i o n , suggesting that i t i s the p r e f e r r e d r e p l i c a t i v e s t r u c t u r e . When l i n e a r pUSVCAT DNA was i n j e c t e d , a very r a p i d conversion to high molecular weight concatemers occurred, r e s t r i c t i o n a n a l y s i s of which revealed a random o r i e n t a t i o n of the l i g a t e d molecules. Some conversion of the l i n e a r molecules to s u p e r c o i l s , 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 r a p i d l y r e p l i c a t e d during cleavage and gastrulation.  I t i s h i g h l y improbable that the 'appearance' of the three  c i r c u l a r forms r e s u l t e d from r e p l i c a t i o n of trace amounts already present i n the stock DNA s o l u t i o n ; some s u p e r c o i l s and open c i r c l e s were already d e t e c t i b l e i n embryos analysed w i t h i n 5 min of t h e i r i n j e c t i o n w i t h l i n e a r pUSVCAT (see F i g 6b, lane 1 ) , and Southern b l o t h y b r i d i z a t i o n of l i n e a r pUSVCAT standards containing the equivalent of one to four times the i n j e c t e d amount showed no evidence of these c i r c u l a r forms (data not shown). The r e s u l t s obtained with supercoiled and l i 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 M a r i n i et al. (1988) i n the Xenopus embryo but d i f f e r somewhat from the processes occurring i n the sea u r c h i n embryo. In the sea u r c h i n embryo, supercoiled plasmids n e i t h e r l i g a t e nor r e p l i c a t e and only l i n e a r molecules are r a p i d l y and e f f i c i e n t l y assembled i n t o end-to-end concatenates (McMahon et al., 1985).  In a d d i t i o n , l i n e a r molecules i n j e c t e d  i n t o sea u r c h i n embryos are not able to reform s u p e r c o i l s or c i r c u l a r molecules (McMahon et al., 1985).  M a r i n i 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 e x h i b i t a preference f o r head-to-tail orientation.  A s i m i l a r mechanism may e x i s t i n the medaka embryo,  52  which could e x p l a i n why r e s t i c t i o n a n a l y s i s of the l i n e a r plasmid-derived concatemers y i e l d e d more of the fragment expected from both u n i d i r e c t i o n a l and random l i g a t i o n s and l e s s of the fragments only expected from random ligations.  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 t e r m i n i .  A Xenopus ovary  s p e c i f i c p r o t e i n 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 u r c h i n e a r l y embryo s p e c i f i c DNA l i g a s e that acts on l i n e a r DNA w i t h cohesive or b l u n t ends (Prigent et al., 1987) have been isolated.  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 u n i t length l i n e a r DNA.  The e x c l u s i v e l y head-  to - t a i l o r i e n t a t i o n of concatemers generated by s u p e r c o i l e d DNA i n d i c a t e that, apart from random l i g a t i o n , another mechanism r e l a t e d to homologous recombination p o s s i b l y e x i s t s i n the medaka embryo, as p o s t u l a t e d f o r Xenopus embryos (Marini et al., 1988).  Folger et al. (1982) have shown i n mammalian  c e l l s that i n j e c t e d l i n e a r or s u p e r c o i l e d plasmids are organized into tandem h e a d - t o - t a i l arrays through homologous recombination between plasmid molecules.  The presence of high molecular weight forms a f t e r 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 r a p i d conversions of the s o r t observed with plasmid DNA  injections  occurred when medaka eggs were i n j e c t e d with e i t h e r CAT phage p a r t i c l e s or CAT phage DNA.  Instead, an upward s h i f t c o n s i s t e n t with a change i n t o higher  molecular weight structures was obvious only from the one-week o l d embryonic stage onwards.  Heating of the embryo DNA e x t r a c t s at 65 °C followed by rapid  c o o l i n g p r i o r to g e l electrophoresis and Southern b l o t a n a l y s i s d i d not produce a band corresponding to CAT phage DNA monomers. I t i s p o s s i b l e that the l i n e a r phage molecules were c o v a l e n t l y l i g a t e d i n t o concatemers e i t h e r w i t h or without m o d i f i c a t i o n of t h e i r cohesive ends.  Covalent end-to-end  53 l i g a t i o n of a recombinant into  A clone has been observed a f t e r  f e r t i l i z e d mouse e g g s A comparison of  (Costantini  the extent  and L a c y ,  form.  F o r eggs  relatively possibly  conversion of  input  T h i s might be  w h i c h appear to be the p r e f e r r e d  increases  post-injection),  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 m i g h t be e x p l a i n e d b y a s u b s e q u e n t  a n d e n s u i n g DNA r e p l i c a t i o n .  significantly  this  stage,  n o t as e x t e n s i v e l y  release  stage  o f DNA f r o m  t o b e no  observations  embryos.  The l a c k  further  and f i s h  the  of a protective for  The i n p u t CAT p h a g e DNA w a s ,  30 s o m i t e n e u r u l a s t a g e ,  30  phage the  supercoiled or  r e d u c e d exogenous  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  ( Z h u e t al.,  1986),  1981).  a similar  i n j e c t e d l i n e a r p l a s m i d DNA h a s b e e n of  i n p u t DNA s e q u e n c e s  c o n t i n u a l l y b e i n g degraded and s u b s e q u e n t l y remained extra-chromosomal  DNA.  stage  throughout  In sea urchins stage  specific  linear plasmid  s u p e r c o i l e d a n d l i n e a r p l a s m i d DNA h a s  ( R u s c o n i and S c h a f f n e r ,  the m a j o r i t y  however,  embryos d e r i v e d f r o m  contained s i g n i f i c a n t l y  o f exogenous  o b s e r v e d i n Xenopus  the  amounts r e m a i n i n g u n c h a n g e d e v e n a t  r e p l i c a t e d a s i n p u t p l a s m i d DNA o f  By t h e  (10 h r  d e g r e e o f DNA r e p l i c a t i o n a n d d e g r a d a t i o n a s c o m p a r e d t o  a n d p h a g e DNA i n j e c t i o n s  loss  blastula  i n j e c t e d p u r i f i e d CAT p h a g e DNA p r o b a b l y a c c o u n t s  greater  conformation.  to be r e l e a s e d from the  There appears  (72 h r p o s t - i n j e c t i o n ) .  CAT p h a g e p a r t i c l e - i n j e c t e d  Since  replicative  (4 h r  coat around the  of  high  u n c h a n g e d up t o t h e e a r l y h i g h b l a s t u l a  somite neurula stage  1985)  into  CAT DNA was  i n DNA a f t e r  subsequent  plasmid  expected  l i n e a r molecules  s u g g e s t i n g t h a t m o s t o f t h e p h a g e DNA h a s y e t  phage c o a t s  decrease  is used.  degree o f  total  post-injection)  al.,  greater  linear  i n j e c t e d w i t h CAT p h a g e p a r t i c l e s ,  phage c o a t s .  Similar  1981).  to a s l i g h t l y  DNA r e p l i c a t i o n when t h e l i n e a r m o l e c u l e  molecular weight concatemers,  injection  o f r e p l i c a t i o n between s u p e r c o i l e d and  p l a s m i d DNA i n j e c t e d e m b r y o s p o i n t s  c o n s i d e r i n g t h e much q u i c k e r  pronuclear  and been  (McMahon e t increase  reported. t h a t were r e p l i c a t e d  lost,  it  embryogenesis.  is  likely  that  Presumably,  were they  extra-  and  54 chromosomally r e p l i c a t e d f o r e i g n DNA  i s unequally segregated among the  daughter c e l l s of a r a p i d l y d i v i d i n g 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 . DNA  In a d d i t i o n , exogenous  segregated i n t o a c t i v e l y d i v i d i n g c e l l s of r a p i d l y growing tissues would  continue to be r e p l i c a t e d , w h i l s t 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 t i s s u e s would be q u i c k l y degraded and  lost.  The net r e s u l t would be an animal mosaic f o r the presence of the f o r e i g n Mosaicism has been demonstrated i n Xenopus where pSV2CAT DNA  DNA.  i n j e c t e d into  f e r t i l i z e d eggs p e r s i s t e d i n tissues of adult frogs but e x h i b i t e d a mosaic p a t t e r n of d i s t r i b u t i o n ( E t k i n and Pearman, 1987). have also shown by d i r e c t in situ  DNA  Hough-Evans et al.  (1988)  h y b r i d i z a t i o n that exogenous CAT  DNA  sequences i n j e c t e d i n t o f e r t i l i z e d sea u r c h i n eggs are mosaically d i s t r i b u t e d 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 r e s u l t a n t mosaicism of non-chromosomal exogenous DNA e x p l a i n the greater CAT l o s s of CAT  may  expression v a r i a t i o n s among the medaka h a t c h l i n g s  and  enzyme a c t i v i t y i n many free-swimming f i s h .  Despite the f a c t that most of the input and r e p l i c a t e d DNA  remain extra-  chromosomal, the p o s s i b i l i t y e x i s t s that genomic i n t e g r a t i o n of some of the exogenous sequences occurred. i n j e c t e d CAT  In t h i s present study, i n t e g r a t i o n of  the  plasmid and phage sequences i n t o 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 i n h e r i t e d CAT i n pooled o f f s p r i n g of several DNA  and phage-treated f i s h .  gene expression  Since germline-  p o s i t i v e 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 a f f e c t the a b i l i t y of a gene to be i n h e r i t e d by Neither does packaging of DNA  offspring.  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 f o r the i n t e g r a t e d sequences.  This  would be expected i f the assumption i s made that the i n c o r p o r a t i o n of the f o r e i g n sequences d i d not occur immediately a f t e r i n j e c t i o n , and that the exogenous DNA was germ c e l l s .  incorporated i n t o only one or a few of several p r i m o r d i a l  In the medaka, Gamo (1961) determined that no p r i m o r d i a l germ  c e l l s could be d i s t i n g u i s h e d p r i o r to g a s t r u l a t i o n , but that up to 20 such c e l l s could be determined at the e a r l y g a s t r u l a stage (15 hr postf 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 i n t e g r a t i o n  at an e a r l y 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 p r o b a b i l i t y that a l l the p r i m o r d i a l germ c e l l s contain the integrated f o r e i g n sequence. The observation that some of the t e s t e d f i s h were not germline p o s i t i v e 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 t i s s u e s . Genomic i n t e g r a t i o n of exogenous DNA  sequences a f t e r i n j e c t i o n i n t o the cytoplasm of f e r t i l i z e d sea u r c h i n egg  has been demonstrated by F l y t z a n i s et a l . (1985).  In a d d i t i o n , germline  transmission of cloned genes a f t e r m i c r o i n j e c t i o n i n t o the egg cytoplasm has r e c e n t l y been reported i n Xenopus ( E t k i n and Pearman, 1987) (Guyomard et a l . , 1988;  and i n f i s h  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 f o r the exogenous sequences, and mosaicism of the founder animals was  i n f e r r e d from the observation that only a  percentage of the o f f s p r i n g from each founder were p o s i t i v e f o r the presence of the exogenous sequences.  I t should be noted that germline transmission p e r  se does not prove s t a b l e host genome i n t e g r a t i o n , as was shown i n the nematode i n which an episomal s t r u c t u r e was passed to the o f f s p r i n g (Stinchcomb et a l . , 1985).  Thus, genomic i n t e g r a t i o n has to be f u r t h e r substantiated by analysis  of Mendelian inheritance of the transgene.  Stuart et a l . (1988) were able to  demonstrate stable i n t e g r a t i o n of a l i n e a r plasmid i n the z e b r a f i s h genome, showing that 50% of F 2 progeny issued from a f o r e i g n DNA p o s i t i v e F-^ outcrossed to an untreated f i s h c a r r i e d the f o r e i g n 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 r a p i d l y tested f o r germline transmission of f u n c t i o n a l CAT  sequences.  Dot b l o t or s l o t b l o t analysis  been employed i n z e b r a f i s h (Stuart et a l . , 1988) used i n t r o u t (Guyomard et a l . , 1988) f o r e i g n DNA  sequences.  However, DNA  detected.  and Southern b l o t analysis  to t e s t o f f s p r i n g f o r the presence of loading constraints f o r d o t / s l o t b l o t s  d i c t a t e the need to load equal amounts of DNA minimum amount of t o t a l DNA  has  w i t h i n a narrow range.  A  i s required i n order f o r s i n g l e copy genes to be  Offspring cannot be pooled since excess t o t a l DNA  may  compete out  any weak h y b r i d i z a t i o n s i g n a l or conversely r e s u l t i n high background s i g n a l s . The DNA  e x t r a c t i o n and h y b r i d i z a t i o n procedures are also comparatively  elaborate.  Southern b l o t a n a l y s i s allows more DNA  to be loaded and thus could  allow pooling of o f f s p r i n g from a s i n g l e parent, but i s comparatively slower than e i t h e r d o t / s l o t b l o t analysis or CAT p o s i t i v e s i g n a l from DNA transmitted CAT  enzyme assay.  Most importantly, a  analysis provides no information on whether the  gene i s f u n c t i o n a l .  Using the simpler and quicker CAT  p r o t o c o l , t e s t s w i t h pooled embryos or hatchlings  assay  from non-treated parents  were negative f o r CAT  a c t i v i t y , and no i n h i b i t o r y e f f e c t could be detected  when a commercial CAT  enzyme was  embryo/hatchling e x t r a c t s .  incubated i n pooled untreated  Thus, not only could F-^ embryos and  hatchlings  from the same DNA/phage treated parent be pooled, but a p o s i t i v e CAT i n d i c a t e d germline transmission of f u l l y f u n c t i o n a l CAT  signal  sequences.  Taken together, the r e s u l t s of t h i s study i n d i c a t e that cloned DNA recombinant phage p a r t i c l e s , c y t o p l a s m i c a l l y  i n j e c t e d at the 1-2  medaka embryos, p e r s i s t and are expressed during embryogenesis.  c e l l stage of No  s i g n i f i c a n t advantage with regard to expression and persistence was when e i t h e r l i n e a r or supercoiled plasmid molecules were used, but apparently more e f f i c i e n t expression was than w i t h the phage DNA  clone.  and  observed an  observed with the plasmid 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 t e s t i n g larger  genes, e s p e c i a l l y genomic sequences containing long stretches of introns or d i s t a n t regulatory regions, which cannot be cloned i n t o plasmids.  In  a d d i t i o n , p r o v i d i n g 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 i n t o 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 e x t r a c t the The very consistent CAT  DNA.  expression s i g n a l s i n the e a r l y embryonic stage  of the medaka strongly favour i t s use as a t r a n s i e n t expression system i n the a n a l y s i s of ' e a r l y ' gene regulatory regions, as has been done, e.g. urchins  ( F l y t z a n i s , et a l . , 1987).  In a d d i t i o n , the medaka embryo can be  i n the f u n c t i o n a l t e s t i n g of genes intended to be used i n the engineering of economically important f i s h e s .  use  genetic  For example, genes coding f o r  the winter flounder a n t i f r e e z e proteins ( L i n and Gross, 1981; 1982;  i n sea  Davies et a l . ,  Gourlie et a l . , 1984), salmon beta-gonadotropin (Trinh et a l . , 1986),  and rainbow t r o u t growth hormone (Agellon et a l . , 1988)  have been cloned.  Introduction of such genes i n t o 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 c o l d water tolerance, f e r t i l i t y , and growth r a t e , r e s p e c t i v e l y . a b i l i t y of the exogenous DNA  The  sequences to p e r s i s t i n some free-swimming stage  medaka, and the p o s i t i v e 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 c r e a t i n g transgenic l i n e s of medaka f o r the study of c e l l lineages and  'late'  gene r e g u l a t i o n , among others. The embryo i s w e l l s u i t e d f o r these purposes since the r e l a t i v e l y large embryo s i z e (ca 1.5 mm diameter) makes manipulation under low  magnifications  (8x to 50x) p o s s i b l e , the two c e l l s derived from f i r s t cleavage are very w e l l 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 s t u d i e s , the chorion can be made more penetrable by m i l d d i g e s t i o n with proteinase K or pronase without having any e f f e c t on the v i a b i l i t y of the embryos.  Thus, harsh treatments such as  trypsin-urea used to d i s s o l v e the chorion of g o l d f i s h eggs (Yamaha et a l . ,  58  1986) or manipulations such as boring t i n y holes i n t o the chorion of t r o u t 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 a l s o obviates the more d i f f i c u l t task of i n j e c t i n g i n t o the germinal v e s i c l e of the medaka oocyte, a procedure adopted by Ozato et a l . (1986), since i n j e c t i o n s i n t o 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 f o r e i g n sequences.  59 REFERENCES  Agellon, L.B., Davies, S.L., Chen, T.T., and Powers, D.A. (1988). 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