Genetic Engineering || Transgenic Fish for Aquaculture

TRANSGENIC FISH FOR AQUACULTURE

Garth L. Fletcherl and Peter L. Davies2

IOcean Sciences Centre Memorial University of Newfoundland St. John's, Newfoundland, Canada AIC 5S7

2Department of Biochemistry Queen's University Kingston, Ontario, Canada K7L 3N6

INTRODUCTION

This review focuses on the technology currently being applied to produce transgenic fish, fish containing novel gene constructs that were experimentally introduced into their genome. A number of excellent overviews on this subject have been published in recent years (1-5). Thus rather than repeat what has already been well stated we have, where possible, attempted to examine critically the published results in order to determine the factors that have or have not been established as being important to the efficient production of stable lines of transgenic fish. We hope that this exercise will be useful to researchers currently applying, or contemplating applying, transgenic technology to fish, and that it will assist all of us in the design of experiments that will enable the production of transgenic fish to be as successful as the production of transgenic mice.

The driving force behind the application of transgenic technology to fish is the desire to produce genetically superior broodstocks for food production. As with all fields of applied biology the success of this technology will be judged solely by the ultimate product. Thus in order to be successful the scientific community must work closely with the aquaculture industry fully to understand its problems, both from a production and marketing point of view. There is little point in promoting the value of the technology if the product can never be marketed.

We came into the field of transgenics some eight years ago in response to a very practical problem facing the aquaculture industry. During the winter the marine environment along most of the Atlantic coast of Canada is characterized by subzero (0 to -1.8°C) water temperatures and the frequent occurrence of surface ice. Salmonids, and most other commercially important fish, freeze if they come into contact with ice at temperatures below -.07°C (6). Therefore sea-cage Genetic Engineering. Vol. /3 Edited by J.K. Setlow, Plenum Press, New York, 1991 331

332 G. J. FLETCHER AND P. L. DAVIES

culture is almost entirely restricted to relatively small areas at the southern edge of eastern Canada where the occurrence of freezing conditions is rare (7,8). Since we had been studying the basis of freezing resistance in fish for many years it seemed reasonable and desirable to respond to the needs of a fledgling industry (9). Since we had already isolated antifreeze protein genes from a freeze-resistant fish, could we transfer them to Atlantic salmon? Can we produce freeze-resistant fish (salmon, trout, charr, halibut, etc.) and thus facilitate the development of aquaculture in northern regions where the only limiting factor is the winter freezing temperatures? In Newfoundland the development of aquaculture enterprises could have a significant impact on the economic viability of many communities scattered along most of the coastline.

Progress in the application of transgenic technology to fish aquaculture has been very rapid, with outdoor performance tests of rapidly growing transgenic carp (Cypdnus carpio), containing a rainbow trout growth hormone gene construct, being carried out within four years of the first published reports on the successful production of transgenic fish with cloned genes (10-13). The reason for this advanced stage of development of transgenic fish compared with transgenic farm animals is largely attributable to the relative ease with which such research can be carried out on fish.

Fish, in contrast to mammals, produce large numbers of eggs (hundreds to thousands) (Table 2) and in many species, particularly salmonids, these eggs can be readily obtained during the reproductive season by gently squeezing (stripping) them out of the genital pore. In addition the eggs of most species under study are very robust and relatively large, ranging from I mm to 7 mm in diameter (Table 2). Their large size and general robustness simplifies handling and microinjection procedures, all of which can be carried out without the necessity of sterile conditions. Since fertilization of the eggs is external it can be delayed for a considerable period of time following egg collection. For example, salmonid eggs and sperm can be transported large distances and fertilization can be delayed for days without significant changes in their viability. Cultivation of the fertilized eggs is, in most cases, very easy, all that is required is an adequate water supply.

WHAT CAN TRANSGENICS DO FOR FISH CULTURE?

It is evident that the potential economic benefits of transgenic technology to aquaculture are paramount. The isolation and construction of genes responsible for desirable traits, and their transfer to broodstocks, could provide a quantum leap over traditional selection and breeding methods. In addition, new traits not present in a genome can be transferred to it from unrelated species, enabling the production of new phenotypes.

There are many changes in the genetic make-up of fish that have been identified as desirable (Table 1). However, for the present, most of these changes can only be regarded as items on a "wish" list, because we are many years from identifying the responsible genes.

At the present time the only available genes that have a high potential value to aquaculture are the growth hormone (GH) and fish antifreeze protein (AFP) genes. The growth-promoting effects of parenterally administered mammalian and piscine GH on fish have been well documented (14-25). Similarly, intraperitoneal injections of purified winter flounder AFP have been

TRANSGENIC FISH FOR AQUACULTURE

Table 1 What Can Transgenic Technology do for Fish

Improve economics of fish culture - increase growth rates - increase overall size - increase dress-out percentage - improve feed conversion efficiencies - utilize low cost diets (carbohydrates as opposed to protein) - improve cold tolerance - improve freeze resistance - improve disease resistance - increase brood stock fecundity - control smolting and reproduction - reduce aggression

2 Tailor fish for the market - external appearance; food fish or exotic tropicals - flesh color, flavor, texture - fatty acid composition

3 Fish as bioreactors - production of medically important compounds - production of commercially useful non-medical compounds

333

4 Basic research aimed at understanding developmental, growth and reproductive processes in fish.

shown to increase the freezing resistance of rainbow trout (26). In addition these AFP can also improve the cold hardiness of plant tissues (27). Since the administration of GH and AFP can produce the desired phenotypic effects in fish it remains to be determined whether the genes coding for these proteins can be stably integrated into the genome and expressed at levels appropriate to their function.

In terms of expression, the antifreeze and growth hormone genes present two very different problems to the investigator. In order to be effective the antifreeze genes must express large quantities of protein that are secreted into the extracellular space to build up levels of 5 to 20 mg/ml (28). Since antifreeze genes are normally expressed in the liver of fish possessing them it is theoretically appropriate to transfer the entire structural gene, including the regulating sequences, and obtain adequate expression. However antifreeze genes are usually present as large multigene families containing up to 150 copies and in addition there is a strong correlation between gene copy number and the level of antifreeze protein expressed in natural populations of fish (95,96). Thus, if only a few gene copies are integrated into the transgenic fish the level of expression may not be sufficient to improve its freeze resistance. The low levels of expression that we have observed to date in our salmon, transgenic for winter flounder AFP gene,


suggest that this may indeed be the case (29). The solution to this problem may be in identifying a sufficiently strong promoter/enhancer, or engineering a protein that is resistant to normal enzymatic degradation.

In contrast to antifreeze proteins, blood growth hormone levels in fish are very low, typically less than 50 nglml. Therefore it may not be necessary for the gene to have a strong promoter. However, it is necessary to modify its tissuespecific expression, because there is little value in transferring GH genes if they can only be expressed in the pituitary gland under homeostatic control via the hypothalamus. In order to avoid this problem investigators construct chimeric GH genes using promoters that would be expected to be expressed in tissues other than the pituitary gland. At the present time promoters include mouse MT-l, RSV and SV 40. In our own research we have coupled the ocean pout antifreeze promoter to the chinook salmon growth hormone (30). A number of groups have reported increased growth rates in fish transgenic for GH (31-33). However the research is still in its infancy, thus it is too early fully to assess the promise and problems posed by the use of the various GH gene constructs.

The potential advantages of transgenic technology for fish research and production can be grouped into the four areas outlined in Table 1. Most current research in the field can be considered under the umbrella of improving the economics of fish culture, a highly justifiable target that has provided the reason for much of the research to be funded. However, although commercial aquaculture is providing the focus for transgenic technology, the research carried out to accomplish this end will generate a basic understanding of gene regulation, developmental biology, physiology and biochemistry of fish in general. This is patently evident in our own research fields, where funds provided by the Strategic Grant Program of the Natural Sciences and Engineering Research Council of Canada to produce freeze-resistant salmon for aquaculture has provided us with the means to examine closely the antifreeze genes and their regulation in the fish that normally possess them (34,35).

What has become evident to all of us is that the successful application of transgenic technology to the production of commercially valuable fish requires the cooperative efforts of molecular and developmental biologists, biochemists, physiologists, geneticists, fish biologists and aquaculture specialists. This bringing together of expertise from all fields of biology to focus primarily on fish as food cannot help but supply the critical mass necessary for the establishment of fish as model vertebrates so eloquently argued for by Dennis Powers (36) and Marcia Barinaga (37).

FISH SPECIES UNDER STUDY

A few characteristics of most of the fish species currently being used in transgenic studies are presented in Table 2. The information contained therein demonstrates the diversity of the species under study, and provides information pertinent to understanding some of the experimental problems posed by their use in transgenic research.

As a result of its applied target, most research on transgenic fish is being carried out on representatives of the world's most important culture species. In an overview of global aquaculture production in 1987, Nash and Kensler (38) present statistics indicating that the cyprinids (carps) (3,939,616 metric tons (t), salmonids

Tab

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F

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Cur

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ly U

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for

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nsge

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Stu

dies

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Sex

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e to

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ge

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ays)

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3-7

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hr

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rout

1-5

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ce/y

ear

800-

1,00

0 3-

5 10

6-

8 hr

-3

5 (

85)

('") c

(83)

r -l

C

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arp

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0.

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90 m

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25

Loa

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47)

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56)

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(salmon and trout) (321,376 t), cichlids (til apia) (246,399 t) and catfish (169,982 t) make up 80% of world production of cultured freshwater fish. The species named in Table 2 approximate 25% of world production.

Apart from tilapia, most of the commercial species are relatively large and have long generation times. Thus they are ill-suited as models to study transgenism. This role appears better suited for tropical species such as the medaka and zebrafish, which are small, relatively easy to culture and have short generation times. One drawback of these small species is the difficulty in obtaining blood samples without killing the fish. At the present time most fish blood growth hormone assays require relatively large plasma samples (50 to 100 /-11). Tilapia species, although larger than the medaka and zebrafish, also have short generation times. Therefore they have potential as model fish and thus provide the researcher with the unique opportunity of coupling basic and applied research on the same species.

GENE TRANSFER TECHNIQUES

To date most of the gene transfer experiments on fish have capitalized on the success of the microinjection techniques developed for mammals. However since fish eggs differ in many respects from those of mammals various details of the injection techniques had to be modified accordingly. In the ensuing sections an attempt is made to evaluate critically various aspects of the microinjection procedures currently being used for fish. In order to do this, techniques to penetrate the chorion, constituents of the injection buffer, injection target and timing relative to fertilization, DNA form and concentration are evaluated somewhat independently of one another. However it is accepted that the successful production of stable lines of transgenic fish in the most efficient way will depend upon an optimum combination of these factors.

Fish Egg Structure

The large size of most teleost fish eggs is due primarily to their quantity of yolk (Figure 1). Upon ovulation and release from the body cavity (spawning) the egg is encased in a thick proteinaceous outer membrane known as the chorion or zona radiata (30 to 40 /-1 thick, salmonids) that serves to protect the egg. This membrane is impermeable to sperm. Therefore all teleost fish eggs that are fertilized following spawning have a single opening (micropyle) in the chorion just large enough to allow the entry of a single spermatozoa. Immediately underlying the chorion, and closely adhering to it in the unfertilized egg, is the vitelline (plasma) membrane. The central part of the egg is filled with yolk while the cytoplasm is present as a thin (~ 40 /-1) peripheral layer that is somewhat thicker (~ 100 /-1) at the animal pole (39).

Meiosis in fishes, as in most chordates, is blocked at the metaphase stage of the second maturation division. The metaphase II plate and the first polar body can be found at the animal pole in close proximity to the micropyle in unfertilized eggs (39,40) (Figure 1). Immediately upon fertilization the egg is activated by contact between the fertilizing sperm and the ooplasm. This activation results in the completion of meiosis and the extrusion of the second polar body under the chorion. At the same time the cortical alveoli, located in the peripheral cytoplasm,

TRANSGENIC FISH FOR AOUACUL TURE

A. UHFERTILIZED UITELLIHE

HEHBRAHE

BLASTODISC

B. FERTILIZED

Figure 1. Teleost egg.

337

discharge their colloidal contents under the chorion resulting in the formation of the perivitelline space and fluid. These changes are accompanied by migration of the peripheral cytoplasm to concentrate at the animal pole, forming the blastodisc (Figure 1). The formation of the fluid-filled perivitelline space allows the developing egg to rotate freely within the chorion. In most eggs the blastodisc remains on top and there is no longer any link between its location and that of the micropyle. In many species the chorion itself becomes a very hard protective shell (39).

Egg Injection

It has been generally assumed that the most effective place to inject gene constructs is directly into the pronucleus or nucleus of the egg before the onset of cleavage. The rationale behind this is that integration into the genome prior to first cleavage will result in all of the organism's cells containing the same copy number of the foreign gene integrated into the same chromosomal sites. Although it is evident that this result can be achieved in mammals (41), available evidence from studies on fish is not as encouraging. The production of mosaics, indicating integration at or after the two-cell stage, appears to be the rule rather than the exception.

One of the difficulties encountered with fish eggs lies with the inability of most investigators to visualize the nucleus or pronucleus using conventional light microscopy. In most cases, if not all, this is attributable to the opaqueness of the chorion and/or the cytoplasm. Rokkones et al. (42) reported unsuccessful attempts to increase the visibility of the pronucleus in salmonid eggs by centrifugation or by vital staining using fluorescent dyes. Centrifugation has proved effective with pig eggs (43). Yamaha et al. (44) have successfully located the female pronucleus in dechorinated goldfish eggs using the fluorescent dye, Hoechst 33342. However no studies have been carried out to determine the effects of this dye, or the ultraviolet light necessary for its fluorescence, on the viability of the eggs. Hammer et al. (43) have used this technique on mammals and report that the procedure damages the ovum.


Since, in most instances, nuclear structures cannot be located, most investigators have opted for cytoplasmic injections following fertilization. Cytoplasmic injections, although not as effective as nuclear injections, have been successful in mice (45). The target for cytoplasmic injections in fish eggs is the thin layer of ooplasm just under the chorion or the developing blastodisc (Figure 1). If the needle penetrates the yolk, the eggs usually die.

In the following sections, the various injection strategies and procedures are described, and their success evaluated on the basis of survival of injected eggs, integration of the exogenous gene into the host DNA, and the degree of mosaicism observed.

Nuclear Injections

Ozato et al. (13) appear to be the only group that have successfully overcome the problem of inserting gene constructs into the nucleus. In order to do this they capitalized on their knowledge of gonadal development in the medaka and surgically removed the oocytes from the ovary prior to ovulation. At the time of removal the cytoplasm is transparent and the large nucleus (germinal vesicle) (120 to 150 Ilm), which is in the prophase of the second meiotic division, is clearly visible and can be readily injected. Following injection, the oocytes are cultured until maturation, fertilized and allowed to develop.

This technique appears to be reasonably successful in that 50% of the injected eggs developed into normal embryos, of which 50% contained the injected sequence (13). However these authors found, using DNA-DNA in situ hybridization techniques, that the nuclei containing the exogenous sequence were distributed mosaicially in every tissue. Thus it appears that injecting fish egg pronuclei does not guarantee integration into all cells.

It is likely that procedures similar to those of Ozato et al. (13) can be developed for other species of fish. However at the present time the additional effort does not appear to be justified, at least for commercial species, because the results obtained to date do not differ appreciably from those reported by others using less sophisticated techniques.

Injection Through Soft Chorions

In fish such as the common carp, channel catfish and loach, the chorion remains relatively soft following fertilization and can be penetrated readily with glass needles (2 to 10 Ilm, carp and catfish, 20 to 35 Ilm, loach) (32,46,47). Dunham et al. (46), and Zhang et al. (32) used as the target for injection the blastodisc prior to cleavage, whereas Korzh (47) injects the yolky interphase immediately adjacent to the blastodisc.

In the catfish, Dunham et al. (46) report an average mortality rate of 87% prior to hatching, compared with <5% for uninjected controls. Powers et al. (62) found a similar mortality rate (- 90%) in their experiments with catfish, although the mortality of their controls was also high (80%). integration rates of the injected sequences, as evidenced by dot blot and genomic Southern blotting procedures, were 20% and 10% for the experiments reported by Dunham et al. (46) and Powers et al. (62) respectively. Neither of these studies present any information on mosaicism.

TRANSGENIC FISH FOR AQUACULTURE 339

Zhang et al. (32) had reasonable success in injecting common carp eggs through the chorion, with mortality rates of injected groups approximating those of controls (~ 65%) and an average genomic integration frequency of 5.5%. These authors also found that integration frequency varied with the stage at which the developing eggs were injected. Injections carried out immediately following fertilization (~ 10 minutes) resulted in approximately 10% of the fish containing integrated sequences. No transgenic individuals were observed in fish developed from eggs injected after this initial period and prior to first cleavage. The highest frequency of genomic integration (15.6%) occurred when injections were carried out during the two-cell stage. The integration frequency at the four-cell stage was 1.4%. Powers et al. (62) in a summary of their research on transgenic fish reported on a similar experiment using catfish eggs. The results were similar to those found by Zhang et al. (32) in that the highest integration frequency (33.3%) occurred when the eggs were injected at the two-cell stage.

Neither Zhang et al. (32) nor Powers et al. (62) provide any information to indicate the degree of mosaicism present in individuals developed from eggs injected at the one-cell stage.

Chong and Vielkind (57) use a combination of reduced water temperatures (12°C) and a Ringer solution to slow down the cleavage rate and hardening of the medaka chorion. They can then penetrate the relatively thin transparent chorion and inject the blastodisc at the one- or two-cell stage. Winkler et al. (65) report that cytoplasmic injections through the chorion at the two-cell stage resulted in a mortality rate of 30% as compared with 22% for uninjected controls. These authors also report that they found some evidence to suggest genomic integration of their gene construct.

Chorion Removal

In many species the chorion becomes very hard once the egg is fertilized, and is a major impediment to microinjection procedures. This problem has been overcome successfully in goldfish, loach and zebrafish, by removing the chorion mechanically or by enzymatic digestion (11,48).

Two groups have reported the successful production of transgenic goldfish by microinjecting the blastodisc of dechorionated fertilized eggs prior to first cleavage (11,60). Yoon et al. (60) reported mortality rates of 90% in their experiments, but noted that in more recent work this had improved to 50%, presumably due to improved technical skills. Zhu (personal communication) finds that mortality rates of injected goldfish, and the eggs of other species he has worked with, depends upon egg quality. With good quality eggs the dechorionation and injection procedures appear to have no effect on mortality rates over that seen for control eggs.

The success of the two groups in terms of integration of the foreign genes into the goldfish genome appears to differ considerably. Zhu et al. (11) report that dot blot hybridization procedures revealed that 50% (3 out of 6) of their 50-dayold goldfish possessed the foreign gene whereas Y oon et al. (60) found only 8% (4 out of 51) of their one-month-old goldfish carried the construct they injected. Evidence from genomic Southern blots indicates that one out of four positive fish had integrated the foreign sequence (60). Zhu et al. (11) could not publish their Southern blots for technical reasons (Zhu, personal communication). However, the restriction patterns they report for the one fish analyzed is consistent with genomic

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(29,

50)

unpu

blis

hed

data

(3

0)

unpu

blis

hed

data

(7

8)

(42)

(4

2)

}>

::;; m

Ul

-t

:D

Tab

le 3

(co

ntin

ued)

» z

Tra

nsge

nic

Fish

Sw

nmar

y en

R

ainb

ow T

rout

G

) m

Z

()

Tem

pera

ture

5-

10

5-10

10

10

10

10

10

"T

1 (0

C)

en C

hori

on s

olut

ion

cut

hole

cu

t ho

le

cut

hole

cu

t ho

le

cut

hole

cu

t ho

le

cut

hole

::c

"T

1 S

ite

inje

cted

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

0

Dev

elop

men

t I

cell

> 2

hr

----

>

I ce

ll 2-

6 h

r --

-->

1

cell

3-5

hr

----

>

----

>

:D

stag

e af

ter

fert

iliza

tion

afte

r fe

rtili

zatio

n af

ter

fert

iliz

atio

n » p

Nee

dle

diam

eter

10

J.l.m

10

J.l.m

10

J.l.m

1

0ll

m

10J.

l.m

10 J

.l.m

10J.l

.m

C

Vol

ume

inje

cted

10

nl

10 n

l 20

nl

20 n

l 20

nl

20 n

l 20

nl

» B

uffe

r 10

mM

Tri

s H

CI

<----

----

10 m

M T

ris

HC

I 50

mM

NaC

l 1

mM

ED

T A

--

----

-->

()

1 m

M E

DT

A,

pH 8

.0

c r D

NA

-t

C

C

onst

ruct

hG

H

hGH

hG

H

hGH

hG

H

rGH

hG

H

:D

Pro

mot

er

mou

se M

T

mou

se M

T

SV

40

SV40

m

ouse

MT

m

ouse

MT

S

V40

m

For

m (

vect

or)

circ

ular

(ye

s)

line

ar (

no)

circ

ular

(ye

s)

line

ar (

yes)

li

near

(ye

s)

line

ar (

yes)

li

near

(ye

s)

Len

gth

(kb)

6.

3 2.

5 6.

6 3.

95

Con

c. (

J.l.g

lml)

500

500

10

10

10

10

25

Inje

ct c

opy

no.

1 x 10

9 (5

000)

Ix

109

(500

0)

-(2

00)

-(2

00)

-(2

00)

3x10

7 (2

00)

Ix10

8 (5

00)

(pg)

%

Sur

viva

l 30

%/-

300/

01-

92%

/100

%

72%

/100

%

60-8

0%/>

90%

60

-80%

/>90

%

20%

/>90

%

inj/

cont

(ag

e)

(hat

ch)

(hat

ch)

(hat

ch)

(hat

ch)

(hat

ch)

(hat

ch)

(hat

ch)

"Int

egra

tion

" %

75

%

75%

40

%

75%

40

% (

2-40

) 38

% (

2-40

) 74

% (

0.5-

50)

(Cop

y N

o.)

(73

d)

(73

d)

(hat

ch)

(hat

ch)

(1 y

r)

(6 m

o)

(6 m

o)

Mos

aic

yes

yes

F 0

expr

essi

on (

%)

72%

mR

NA

, 72

% m

RN

A,

no

no

no

14%

GH

42

% G

H

(em

bryo

) (e

mbr

yo)

Inhe

rita

nce

F /F

2 (%

) 16

%/-

29,8

,12,

20%

/-

Exp

ress

ion

F/F

2 no

no

(%

) R

efer

ence

(4

2)

(42)

(5

3)

(53)

(5

9)

(59)

(5

9)

-~.--

CAl ~

w ~

I'J

Tab

le 3

~co

ntin

ued)

T

rans

geni

c is

h S

umm

ary

Rai

nbow

Tro

ut

Loa

ch

Tem

p'er

atur

e (0

C)

9.5

9.5

9.5

21

Cho

rion

sol

utlO

n st

rong

nee

dle

stro

ng n

eedl

e st

rong

nee

dle

soft

cho

rion

S

ite

inje

cted

pe

rivI

tell

ine

spac

e bl

asto

disc

bl

asto

disc

bl

asto

disc

-yol

k

Dev

elop

men

t st

age

afte

r fe

rtil

izat

ion

imm

edia

tely

im

med

iate

ly

boun

dary

0.

5-1

hr

afte

r fe

rtil

izat

ion

afte

r fe

rtil

izat

ion

afte

r fe

rtil

izat

ion

Nee

dle

diam

eter

<15~m

< 1

5 ~m

< 1

5 ~m

20-3

0 ~m

Vol

ume

inje

cted

18

nl

18 n

l 18

nl

10 n

l B

uffe

r 10

mM

Tri

s H

Cl

<--

----

10 m

M T

ris

HC

l 0.

125

----

-->

88 m

M N

aCI

pH 7

.5

mM

ED

TA

pH

7.5

DN

A

Con

stru

ct

rGH

rG

H

rGH

pA

T15

3 P

rom

oter

m

ouse

MT

m

ouse

MT

m

ouse

MT

F

orm

(ve

ctor

) li

near

(ye

s)

line

ar (

no)

circ

ular

(ye

s)

circ

ular

and

lin

ear

(yes

) G

') L

engt

li (

kb)

6.6

6.6

8.9

3.6

C.o

nc. (~g/ml)

0.4

6 0.

4 6

0.5

6 50

!-

Inje

ct c

9PY

. :p.~

. (p

gl

Ixl0

~7~

lxlO

~7)

lxl0

&

10)

lxl0

8 (5

00)

"T1

% S

urvI

val

illJ/

con

57%

/4 -

6%

25%

/4 %

32

%/5

%

r m

(age

) &

hatc

h)

~ale

vins

+ fr

y)

~ale

VinS

~ -f

"Int

eera

tion

" -4

% (

1)

% (

>1)

%

(>1

5

%

n J:

% (

.op

y N

o.)

(fry

) (f

ry)

(ale

vins

(3

0 d)

m

:lJ

M

osaI

C

yes

~

F ex

pres

sion

J;(o)

z I&

hert

tanc

e F

J 2 ~~

0 E

XR

ress

ion

F /F

2 Yo

:0

R

e er

ence

(5

2)

(58)

(5

8)

(76)

r- 0 ~

:5

m

en

-I

JJ

}>

Tab

le 3

(co

ntin

ued)

Z

T

rans

geni

c Fi

sh S

umm

ary

en

c;) m

Z

Com

mon

Car

p C

atfi

sh

Nor

ther

n Pi

ke

n J:!

en

J:

Tem

pera

ture

(o

q

18-2

0 18

-20

26-2

7 20

20

20

"T

1

Cho

rion

sol

utio

n so

ft

soft

so

ft

soft

so

ft

pene

trat

e w

ith

0 JJ

need

le

}>

Site

inj

ecte

d bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

bl

asto

disc

p

Dev

elop

men

t st

age

1-4

cell

1-

4 ce

ll 1

cell

5-9

0 m

in

1-4

cell

1-

4 ce

ll

1 ce

ll 1

0+ m

in a

fter

c

afte

r fe

rtili

zatio

n fe

rtili

zatio

n }>

("

) N

eedl

e di

amet

er

2-10

11m

2-

10 1

1m

2-10

11m

2-

10 1

1m

2-10

11m

2-

3 11

m

c V

olum

e in

ject

ed

20 n

l 20

nl

20 n

l 20

nl

20 n

l 2

nl

r -I

Buf

fer

10 m

M T

ris

HC

l 10

mM

Tri

s H

Cl

c 1

mM

ED

TA

88

mM

NaC

l JJ

m

D

NA

C

onst

ruct

R

ainb

ow t

rout

hG

H

hGH

co

ho G

H c

DN

A

Rai

nbow

tro

ut

bGH

cD

NA

G

HcD

NA

G

H c

DN

A

Pro

mot

er

RS

V-L

TR

m

ouse

MT

m

ouse

MT

R

SV

-LT

R

RS

V-L

TR

R

SV

For

m (

vect

or)

line

ar (

yes)

li

near

(ye

s)

line

ar (

no)

line

ar (

yes)

li

near

(ye

s)

circ

ular

(ye

s)

Len

gth

(kb)

5.

2 6.

3 2.

9 5.

5 5.

2 C

onc.

(lt

g/m

l)

0.25

0.

35

0.15

0.

5 0.

3 12

.5

Inje

ct c

opy

no.

(pg)

lx1

06 (

5-6)

1x

106

(7)

1x10

6 (3

) lx

106

(10)

1x

106

(6)

-(2

5)

% S

urvi

val

injl

cont

37

%/3

4%

40%

13

%/>

95%

9.

5%/2

0%

5%/-

(age

) (h

atch

) (h

atch

) (h

atch

) (h

atch

) (h

atch

) "I

nteg

rati

on"

%

5.5%

(2-

10)

5%

20%

(1-

2)

10%

(2-

20)

6%

5%

(Cop

y N

o.)

(90

d)

(90

d)

(3 w

k)

(8-1

0 w

k)

(90

d)

(7 m

ol

Mos

aic

yes

F 0

expr

essi

on (

%)

5.5%

(G

H,r

bc)

4% s

erum

bG

H

(90

d)

(als

o gr

owth

) In

heri

tanc

e F

/Fl (

%)

0,32

,42,

100/

-E

xpre

ssio

n F

/Fl (

%)

30-4

0% l

arge

r/-

(46)

R

efer

ence

(3

2)

(62)

(6

2)

(62)

(3

3)

w

.j:> w

Tab

le 3

(co

ntin

ued)

w

~

Tra

nsge

nic

Fis

h S

umm

ary

~

Med

aka

Tem

pera

ture

26

(D

C)

26

25

25

25

26

26

Cho

rion

sol

utio

n pr

e-ov

ulat

ion

pre-

ovul

atio

n R

inge

r to

kee

p R

inge

r to

kee

p R

inge

r to

kee

p pr

e-ov

ulat

ion

e1ec

trop

orat

ion

soft

so

ft

soft

S

ite

inje

cted

oo

cyte

nuc

leus

oo

cyte

nuc

leus

bl

asto

disc

bl

asto

disc

bl

asto

disc

oo

cyte

nuc

leus

D

evel

opm

ent

pre-

ovul

atio

n pr

e-ov

ulat

ion

1-2

cell

1-2

cell

1-2

cell

1 ce

ll st

age

Nee

dle

diam

eter

3-

4 J.l

m

3-5

J.lm

3-

5 J.l

m

3-5

J.lm

3-

4 J.l

m

Vol

ume

inje

cted

20

-30

pi

500

pi

500

pi

500

pi

30 p

i B

uffe

r 0.

1 m

M T

ris

HC

l +

0.2

5%

+ 0

.25%

0.

1% p

heno

l sa

line

0.

01 m

M E

DT

A

phen

ol r

ed

phen

ol r

ed

red

DN

A

Con

stru

ct

chic

ken

cS ft

refl

y lu

cife

rase

C

AT

C

AT

C

AT

ra

inbo

w t

rout

ra

inbo

w t

rout

cr

ysta

llin

G

HcD

NA

G

HcD

NA

P

rom

oter

R

SV

LT

R,

SV40

L

TR

, S

V40

C

MV

, th

ymid

ine

mou

se M

T

mou

se M

T

kina

se

Form

(ve

ctor

) ci

rcul

ar (

yes)

ci

rcul

ar a

nd

line

ar C

AT

ci

rcul

ar (

yes)

li

near

(ye

s)

linea

r (y

es)

linea

r (y

es)

phag

e D

NA

L

engt

h (k

b)

14.4

7.

9 44

.2

5.2

5.2

Con

c. (

J.Lgl

ml)

10

50

20

50

1.0

G)

Inje

ct c

opy

no.

103 _1

04 (0

.2-0

.3)

3xlO

6 (2

5)

2x10

5 (1

0)

(25)

2x

lO6

(10)

c...

. (p

g)

-n

% S

urvi

val

48%

/-72

%/-

32%

/-25

%

r in

j/co

nt (

age)

(7

d)

(hat

ch)

(hat

ch)

m

-i

"Int

egra

tion

" %

50

% (

1-10

0)

29%

/-20

-30%

/ 25

%/-

27%

(1-

100)

4%

(1-

100)

()

:r:

(Cop

y N

o.)

(6 d

) (I

mo)

(3

mo)

(h

atch

) (h

atch

) m

M

osai

c ye

s ye

s ::I

l

Fa e

xpre

ssio

n (%

) 31

% (

cS cr

y.)

29%

/-20

% (

CA

T)

50%

(C

AT

) ye

s » z

(7 d

) (6

d)

(I m

o)

(l m

o)

0 In

heri

tanc

e F

/F2

100%

/88%

:u

(%)

r E

xpre

ssio

n F

/F2

0 (%

) »

Ref

eren

ce

(13)

(9

4)

(57)

(5

7)

(65)

(7

4)

(74)

<

m

(f

)

Tab

le 3

(co

ntin

ued)

T

rans

geni

c Fi

sh S

umm

ary

-I

::D »

Zeb

rafi

sh

Tila

pia

Gol

dfis

h z (J

) G

) m

Z

Tem

pera

ture

28

28

27

25

20

-25

21

21

n (0

C)

"'T1

Cho

rion

sol

utio

n re

mov

e re

mov

e m

icro

pyle

st

rong

nee

dle

rem

ove

rem

ove

rem

ove

en I Si

te i

njec

ted

blas

todi

sc

blas

todi

sc

blas

todi

sc

blas

todi

sc

blas

todi

sc u

nder

bl

asto

disc

bl

asto

disc

"'T

1 po

lar

body

0

Dev

elop

men

t 1-

4 ce

ll

1 ce

ll 1 +

c~l

ls

1 ce

ll 1

cell

1 ce

ll 1

cell

::D »

stag

e p

~eedle d

iam

eter

3

Itm

3

Itm

5

ltm

2

5lt

m

3-41

tm

2lt

m

2lt

m

C

Vol

ume

inje

cted

30

0 pI

30

0 pI

1-

2 nl

20

nl

1-2

nl

2 nl

2

nl

» B

uffe

r 2%

phe

nol

red

0.5%

phe

nol

red

10 m

M T

ris

HC

l sa

line

10 m

M T

ris

HC

l --

-->

--

-->

n c

0.2

mM

ED

TA

88

mM

~aCI p

H

+ b

lue

dye

+ b

lue

dye

r pH

8.0

7.

5 -I

c

D~A

::D

Con

stru

ct

E. c

oli h

ygro

C

AT

hG

H

bGH

hG

H

CA

T

m

neo

Pro

mot

er

SV

40

RSV

,SV

40

mou

se M

T

LT

R

mou

se M

T

RSV

R

SV

For

m (

vect

or)

line

ar (

yes)

ci

rcul

ar (

yes)

li

near

(ye

s)

linea

r (y

es)

line

ar (

yes)

li

near

(ye

s)

circ

ular

(ye

s)

Len

gth

(kb)

5.

2 4.

0 8.

5/3.

8 9.

4 5.

3 C

onc.

(lt

g/m

l)

100

160

6-12

5-

~50

27

25

25

Inje

ct c

opy

no.

6x

l06

(30)

-

(50)

Ix

106

(12)

10

_10

8 7x

l06

(20-

40)

0.5x

1 06

(50)

-

(50)

(p

g)

(100

-500

0)

% S

urvi

val

16%

/-80

%17

5%

5%/3

8%

10-5

0%/9

0%

10-5

0%/9

0%

inj/c

ont

(age

) (1

0 d)

(h

atch

) (h

atch

) (1

mol

(1

mol

"I

nteg

ratio

n" %

5%

(1-

100)

18

%

6%

30%

50

%

7% (

>1)

60

%

(Cop

y ~o

.)

(4 m

ol

(4 m

ol

(90

d)

(adu

lt)

(50

d)

(1-2

mo)

(>

1 m

ol

Mos

aic

yes

yes

yes

yes

Fo e

xpre

ssio

n (%

) no

18

% (

CA

T)

mR~A

70%

(C

AT

) (4

mol

(1

-2 m

ol

(> 1

mol

In

heri

tanc

e F

/F2

20%

/50%

F\

36,

54,1

2,25

(%

) F2

53,

48,5

6,53

E

xpre

ssio

n F

/F2

yes

(%)

Ref

eren

ce

(48)

(6

3)

(55)

(5

6)

(11)

(6

0)

(33)

to)

-: no

inf

orm

atio

n; -

--->

as

prev

ious

; di

ffer

ent

num

bers

for

F\

and

Fl

refe

r to

dif

fere

nt c

ross

es;

(vec

tor)

ref

ers

to p

rese

nce

or a

bsen

ce.

~

t1I


integration. The high integration frequency found by Zhu et al. (1) may be related to their ability to target the blastodisc nucleus by injecting immediately under the second polar body which could be visualized on the surface of the dechorionated egg (personal communication) (see also Figure 1). Zhu's group also reports high integration frequencies (~ 40%) using the same technique to inject eggs of loach, common carp, red crucian carp, silver crucian carp, mirror carp and red carp (61).

Stuart et al. (48) reported that their method of injecting dechorionated zebrafish eggs at the 1- to 4-cell stage resulted in a mortality rate of 84%. However, as the authors indicate, a significant proportion (~ 30%) of this mortality rate is attributable to the amount of DNA injected rather than the egg injection procedures themselves (see upcoming discussion). These authors (48) also found evidence to suggest that only 5% of their adult (four months) fish retained the foreign DNA and that less than 1 % of the injected fish were germline transformants. Most, if not all, of these transgenic fish (F)l) appeared to be mosaics. Further studies were carried out by Stuart et al. (03), in which they injected the zebrafish eggs prior to first cleavage. In these studies they report evidence to suggest DNA retention rates of at least 19%, with 5% of the surviving fish being germline transformants. However despite the fact that their transformation rates may have improved by confining their injections to the one-cell stage, the transgenic fish were frequently if not always mosaics.

Chorion removal procedures cannot be applied universally. Hallerman et al. (49) reported that although they could dechorionate northern pike eggs using protease type XXV, mortality was high and surviving eggs very fragile. Fragile eggs are not likely to survive microinjection procedures. These authors could not find an effective procedure to dechorionate walleye eggs, enzymatic or mechanical. In our laboratory we have found that Atlantic salmon and brook trout (Sa/ve}inus fontina/is) chorions can be completely, or partially, removed surgically to allow on unimpeded view of the early cleavage stages. However, despite considerable effort, the embryos have not survived longer than a few weeks (Wu, unpublished data).

Injection Through Hard Chorions

Three solutions are currently being used to overcome the hard chorion of salmonids and at the same time target the nuclear areas or blastodisc. We have found that salmonid eggs (Atlantic salmon, rainbow trout, brown trout (Sa/mo trutta), Arctic char (Sa/velinus a/pinus)) can be readily injected through the micropyle using very fine glass needles (2 to 3 ~m external diameter) (50,51, unpublished observations). In addition to facilitating microinjection procedures, the micropyle also provides us with a means of locating the male and female pronuclei. The fertilizing sperm nucleus remains in the ooplasm immediately under the micropyle which in turn is in close proximity to the female pronucleus. Union of the male and female pronuclei does not appear to occur until the eggs are activated by placing them in water. Therefore microinjection through the micropyle in fertilized but non-activated eggs places the gene constructs in very close proximity to the pronuclei.

Maclean et al.'s (52) solution to injecting rainbow trout eggs is to rely on sturdy glass needles (::; 15 ~m) to penetrate the chorion soon after fertilization before the hardening process has been completed. At this time the pronuclei would


Table 4 Relative Success of Salmonid Egg Injection Procedures

Egg Stage Injection Survival Exogenous Mosaics References Procedure (% of controls) DNA

persistence (%)

pre-pronu- micropyle 90-100 1-6 Yes (50) clei fusion 20% inheri-

tance in F\ early one perinuclear 55 0-18 Yes (58,93) cell cytoplasm x=8 mosaic tissues

in Fo late one cell well-developed 70 38-74 Yes (59)

blastodisc x = 50 mosaic tissues in Fo 8-29% inheri-tance in F\

late one cell well developed 30 20-75 (42) blastodisc

Survival values refer to hatching or later stages. The 55% survival presented for reference 58 is an average value computed from all experiments with the early injection procedure. Average uninjected control eggs = 46%; average injected eggs = 25.3%. The mean % fish containing exogenous DNA sequences is also computed (58)

have fused. Marc Welt of Battelle-Pacific uses a procedure similar to that of Maclean except that a 33 gauge steel needle is used instead of a glass one (personal communication). Rokkones et al. (12), Chourrout et al. (53) and Gibbs et al. (54), who are also working with salmonids, wait until the water hardening process has been completed and then cut a small hole in the chorion to allow the insertion of a glass needle (~ 10 /-lm) into the well-developed blastodisc prior to first cleavage.

The success of the three techniques used to inject salmonid eggs is presented in Table 4. Once we had mastered the technique of injecting through the micropyle we found no differences in survival, at hatch or first feeding, between injected and uninjected groups. Survival rates are always high provided the egg quality is good and the water quality adequate. Investigators using the other two techniques report a higher mortality in injected as compared with uninjected controls (Table 4). The high mortality rates reported by Penman et al. (58) suggest that their technique of penetrating the chorion with the injecting needle prior to water hardening is quite injurious. However the high mortality rates (~ 50%) of the control, uninjected groups in their experiments are suggestive of egg and/or water quality problems. If this is the case then any egg injection procedure may cause high mortalities. In our experience egg quality is highly important. It


appears to vary considerably from female to female and deteriorates with time following ovulation and following artificial stripping.

It is evident that all three salmonid egg injection techniques result in a proportion of the fish containing what appears to be genomic integration of the injected DNA sequences (Table 4). Rokkones et al. (42) indicated that 6 out of 8 (75%) 73-day-old embryos and one out of five (20%) one-year-old salmon contained the injected sequence. However they could not determine whether or not they were genomically integrated.

Accepting that the criteria for integration are comparable it would appear that Guyomard et al. 's (59) technique of injecting the well-developed blastodisc prior to first cleavage is the most successful, for they found that approximately 50% of their 6- to 12-month-old rainbow trout contained integrated sequences. Our own technique of injecting through the micropyle prior to fusion of the pronuclei appears to be the least successful (51). All of the transgenic salmonids that have been reported on in detail appear to be mosaics. Thus there does not appear to be any advantage in attempting to inject early in the first cleavage cycle. In fact the high integration frequency rates reported by Guyomard et al. (59) suggest that it may be better to wait until the blastodisc is well developed.

Penman et al. (58) reported the results of a series of experiments aimed at comparing the three salmonid injection techniques. They found no differences between the techniques in terms of genomic integration of the injected DNA. Unfortunately the mortality rates for these experiments were so high (59 to 98%) that it is difficult to be confident that this was in fact the case.

We recently carried out an experiment in our laboratory to determine whether injecting the well-developed blastodisc would result in a higher integration frequency than we were obtaining by injecting prior to pronuclei fusion. In this experiment we allowed the fertilized Atlantic salmon eggs to water harden for 4 to 5 hours before injecting I x 106 copies of our 7.8 kb winter flounder antifreeze gene into the center of the blastodisc through the micropyle. 600 eggs were injected of which approximately 500 survived to first feeding. peR analysis of 50 4- to 5-month-old fry revealed that only one of them (2%) contained the injected sequence (Fletcher and Davies, unpublished data). Thus in our hands the time of injection does not influence the potential integration efficiency. If this result can be generalized it would appear that the high integration frequencies reported by Guyomard et al. (59) must be attributable to some factor other than the timing of their injections, perhaps the amount of DNA injected (see later discussion).

It is of interest to note at this point that it may not be necessary to inject the developing blastodisc in order successfully to introduce gene constructs into salmonids. Marc Welt (Battelle-Pacific) obtains a 7% incorporation frequency by injecting the gene constructs into the perivitelline space of rainbow trout eggs prior to first cleavage (personal communication). This technique was also used with some success by Norman Maclean during his earlier experiments (personal communication). These results indicate that macromolecules can find their way from the perivitelline space into the embryonic cells, possibly due to chance, when the egg cell membrane is undergoing its many biochemical and physical changes during early cleavage prior to completion of the periblast.

Two techniques are being used to penetrate the hard tilapia chorion. Brem et al. (55) have successfully used the micropyle route to inject the blastodisc of fertilized eggs at the 2- to 4-cell stage using a 5 Ilm diameter needle. Phillips (56)


first cools the eggs to 20° C to delay the time to first cleavage and then uses a relatively large, strong needle (25 11m diameter) to penetrate the chorion in the area of the micropyle and inject the single-celled embryo.

The two techniques used to inject tilapia eggs clearly differ with respect to survival. Brem et al. (55) found an average mortality rate of 20% at hatch among eggs injected through the micropyle. Mortality rates for control uninjected eggs were approximately 25%. Phillips (56), who injected the one-celled blastodisc through the chorion using a larger needle, observed mean mortality rates of 90 to 95%. Control mortality rates were approximately 50%. Sham microinjection experiments by Phillips clearly established that the physical act of microinjecting the eggs accounted for the difference in mortality rates between control uninjected and DNA injected eggs.

Both investigations on tilapia report evidence for genomic integration of exogenous sequences. However, Brem et al. (55) found an integration frequency of 6%, while Phillips (56) reported a value of 30%. Neither study reports on information relevant to mosaicism. However, since Brem et al. (55) carried out their injections after first cleavage, all fish would be expected to be mosaic.

DNA Concentration

Most investigators have had no choice but to carry out cytoplasmic rather than nuclear injections of gene constructs. Thus they have opted to inject millions of gene copies in the hope that a few would survive to enter the nucleus and become integrated into the genome. The general strategy is to increase the probability of integration by injecting as much DNA as the egg can take without suffering undue mortality or abnormal development.

The amount of exogenous DNA that can be tolerated by early embryos appears to differ considerably between species. A comparison between zebrafish and medaka with the use of identical techniques indicated that zebrafish are more sensitive to injected DNA with 50 pg being lethal to 84% of the embryos (Table 5) (48,64). In contrast, salmonid embryos appear to be much more tolerant of the injected DNA than the medaka or the zebrafish. Mortality rates for salmonids do not differ appreciably from controls until the amount of injected DNA exceeds 200 pg, and substantial numbers survive amounts as high as 5000 pg (Table 5). This amount of DNA is approximately 103 times the salmonid genome equivalent (3 x 109 bp). The greater amount of DNA tolerated by salmonids is likely related to the fact that their egg volume is some 200 times that of the medaka or zebrafish eggs (Table 2).

Phillips (56) carried out a series of experiments in which fertilized eggs of tilapia were injected with 100, 500, 1000 or 5000 pg of a bovine growth hormone sequence linked with an avian LTR (3.8 or 8.5 kb). Although he found no statistically significant differences in survival to hatch between the groups, there was a trend towards greater mortalities as the amount of DNA injected increased. Overall survival of the DNA-injected group was low (7.5%) and statistical analysis of the data is confounded by the high mortality caused by the injection procedure itself (% survival = 15.6%) and by the highly variable survival rates of uninjected embryos.

One striking feature of the salmonid data presented in Table 5 is that, assuming the criteria for genomic integration are comparable, it appears that the potential to produce transgenic salmonids increases dramatically when the amount

Tab

le 5

E

ffec

ts o

f In

ject

ed D

NA

on

Fish

Em

bryo

Sur

viva

l an

d T

rans

geni

c Fr

eque

ncy

DN

A p

glem

bryo

7

10

12

25

50

70

150

200

250

500

700

5000

D

NA

con

cent

rati

on

(!.tg

lml)

0.4

20

3 50

10

0 4.

0 30

0 10

50

0 25

40

50

0 V

olum

e in

ject

ed (

nl)

18

0.5

4 0.

5 0.

5 18

0.

5 20

0.

5 20

18

10

D

NA

con

stru

ct c

opy

num

ber/

embr

yo

lxl0

6 lx

106

lxl0

6 3x

l06

6x10

6 lx

l07

2x10

7 3x

107

3x10

7 1x

108

lxl0

8 lx

l09

Bas

e pa

irs/

embr

yo

7x10

9 8x

109

8x10

9 2x

lOIO

5x

l0lo

7x

1OIO

1.6x

lOII

2x1O

II

2.4x

l011

4x

1OII

7xl0

11

2.5x

l012

Zeb

rafi

sh (

% s

urvi

val)

43

24

16

0

0 %

Int

egra

tion

5-

18

Med

aka

(% s

urvi

val)

72

38

0

Sal

mon

ids

(% s

urvi

val)

99

90

80

70

20

56

30

%

Int

egra

tion

0

1-6

4 38

74

18

20

-75

(Ref

eren

ce)

(58)

(5

0)

(58)

(5

9)

(59)

(5

8)

(42)

Zeb

rafi

sh a

nd m

edak

a da

ta o

btai

ned

from

ref

eren

ces

48 a

nd 6

4. D

NA

con

stru

ct =

pU

SV

CA

T (

7.9

kb).

Sur

viva

l w

as e

stim

ated

one

wee

k fo

llow

ing

inje

ctio

n. S

alm

onid

dat

a ob

tain

ed f

rom

sev

eral

sou

rces

. 58

: R

ainb

ow t

rout

. D

NA

con

stru

ct =

M

T r

GH

(6.

6 kb

), %

sur

viva

l w

as c

ompu

ted

as f

ollo

ws:

%

egg

s su

rviv

ing

DN

A i

njec

tion

x

100.

50:

Atla

ntic

sal

mon

. D

NA

con

stru

ct =

an

tifr

eeze

pro

tein

, (7

.8 k

b) 5

9: R

ainb

ow t

rout

. D

NA

con

stru

ct =

eggs

sur

vivi

ng b

uffe

r in

ject

ion

seve

ral

incl

udin

g, p

MT

rGH

, (6

.6 k

b) 2

00 p

g; p

SV51

8, (

3.95

kb)

500

pg.

42:

Rai

nbow

tro

ut a

nd A

tlan

tic

salm

on.

DN

A c

onst

ruct

= tw

o in

clud

ing

MT

hGH

(2.

5 kb

), %

sur

viva

l fo

r al

l o

f th

e sa

lmon

id d

ata

was

est

imat

ed a

t ha

tchi

ng o

r la

ter

stag

es.

% i

nteg

ratio

n is

ten

tativ

e.

w

c.n

o G) c....

"T1 r m

-t

()

I m

:::0 ~

Z o :u r o ~

<

m

en


of DNA injected is 200 pg or more (approximately 100 times the normal amount of genomic DNA). For ease of discussion this can be termed the "DNA load dependent-jntegratjon' hypothesis. Support for this hypothesis comes from an experiment by Penman et al. (58), who found that the proportion of rainbow trout alevins containing exogenous sequences increased from 0% to 18% when the injected load of DNA was increased from 7 to 700 pg. In addition Guyomard et al.'s (59) data on six-month-old rainbow trout suggests that the integration frequency increased from 28% to 74% when the DNA load injected increased from 200 to 500 pg (Table 5).

The 18% putative transgenic zebrafish observed in a recent study by Stuart et al. (63) suggests that a relatively high rate of genomic integration can be obtained in this species using considerably less DNA (25 pg) than used in salmonids (Table 5). Salmonid eggs have approximately 200 times the volume of zebrafish eggs and it is likely that they also have a larger volume of yolk-free cytoplasm. Thus although the amounts of injected DNA differ considerably between the two species the final concentrations of exogenous DNA in the egg cytoplasm may be similar.

DNA Form

Very few studies on the production of transgenic fish have included the effects of DNA form in their experimental design. Most investigators appear to have been using linearized as opposed to circular constructs because studies on mice have demonstrated that they are integrated into the genome more efficiently (45). However this fact has not yet been established for fish.

One of the problems faced by investigators using fish is that it is much more difficult clearly to establish genomic integration than it is in mice. Several factors contribute to this difficulty. In the first instance the amount of DNA injected into fish eggs is orders of magnitude greater than that injected into mice eggs: compare the 6 x 106 copies injected into a medaka egg (Table 5) with the few hundred copies injected into mice (45). This amount of DNA injected into fish eggs is, in most cases, equal to or greatly in excess of the amount of DNA normally present in a diploid cell. There is also abundant evidence indicating that the DNA injected into fish eggs (circular or linear) is amplified and that much of it persists extrachromosomally, possibly rearranged, throughout embryogenesis (11,48,52,57). In some cases it appears to persist at least until the fish are sexually mature. Non-integrated DNA is rapidly degraded in mammalian eggs; thus evidence for integration normally only requires the demonstration that it is present in an intact form. The persistence of extrachromosomal DNA in fish has required investigators to resort to genomic Southern blot analysis in an attempt to demonstrate the presence of potential junction fragments between the injected sequence and the genomic DNA. However, as good as some of the evidence appears to be, a general consensus is emerging that it may be impossible to demonstrate integration unequivocally in the injected fish (F 0) and that it will be necessary to wait for the results from F 1 or even F 2 generations.

Two investigations have reported results to suggest that the use of linearized constructs will result in a higher frequency of integration than will supercoiled ones. In an early study Chourrout et al. (53) compared the effects of circular and linearized forms of the plasmid pSV507 when injected into fertilized rainbow trout eggs. They found evidence to suggest that a higher percentage


(75%) of the newly hatched fry injected with the linearized plasmid contained the exogenous gene than did fry injected with the supercoiled fonn (40%). Restriction analysis was not carried out in this study and thus the frequency of genomic integration of either DNA fonn could not be estimated. Survival to hatch was similar in both groups (circular 92%, linear 66%).

Penman et al. (58) also found that the frequency of rainbow trout fry containing the injected sequence was greater in the ones developed from eggs injected with the vector-free linear fonn (MTrGH, 6.6 kb) (11 %) than it was in those injected with the supercoiled fonn (4%) (pMTrGH, 8.9 kb). Moreover these authors concluded from their genomic Southern blot analyses that the majority of the exogenous DNA in the fry injected with the circular plasmid was present as an extrachromosomal fonn rather than being integrated into the genome. Only one (~ 1%) of the fish injected with the circular plasmid was considered to have integrated the DNA. DNA fonn did not affect embryo survival (32% supercoiled, 25% linear, 52% controls). Although these results are consistent with the hypothesis that the frequency of genomic integration is higher with linear compared with circular constructs, they are not convincing. The number of potentially transgenic fish examined was small and three other experiments reported in the same study, using the same linearized construct, indicate integration frequencies (0, 1 and 3%) similar to that found for the circular construct (1 to 4%) (58).

The results reported on a number of other investigations do not indicate that it would be better to linearize constructs prior to microinjection.

Rokkones et al. (42) injected circular and linearized MThGH plasmids into salmonid eggs and although the injected sequences (circular and linear) were present in 20 to 75% of the fish they did not comment on differences between the two constructs in tenns of integration efficiencies. The only transgenic one-yearold Atlantic salmon examined had been injected with the circular construct. These authors did report that the circular construct appeared to be the better template for hGH gene transcription during embryogenesis.

Chong and Vielkind (57) compared the expression and fate of four fonns of the CAT reporter gene microinjected into medaka eggs. They found that all four fonns (supercoiled, linear, phage particles and phage DNA) were replicated and expressed throughout embryogenesis and, in some instances, into early adulthood when the study terminated. The persistence and expression of the injected CAT genes in free-swimming medaka, and the fact that these genes comigrated with high molecular weight DNA as seen in Southern blots, is consistent with genomic integration of some of the exogenous sequences. There were no obvious differences in the retention of the various fonns of the CAT gene.

A recent study (63) demonstrates clearly that stable lines of transgenic zebrafish can be generated with a supercoiled construct (RSV, SV40, CAT). In addition the results of this group also suggest the possibility that circular constructs may be preferable to linearized ones, for the percentage of adults containing the injected sequence was greater than that found in an earlier study where a linear sequence was used (18% vs 5%) (48). Moreover the percentage of germline transfonnants was also greater in the more recent study with circular constructs (5% vs 1 %) (63).

There are other differences between the two studies by Stuart et al. (48,63), gene constructs and time of injection for example. Thus finn conclusions about the relative value of the different DNA fonns are premature.


Injection Buffer

Few studies report on the effects of the buffer used to inject DNA into the eggs. Zhu feels that buffers containing EDT A result in significant mortalities to goldfish and carp eggs. Penman et al. (58) support this contention by reporting that survival of rainbow trout eggs is best when a Tris-NaCl buffer is used instead of ones containing EDT A. We have found that buffers containing I mM EDT A (with or without DNA) can, on occasion, be lethal to Atlantic salmon embryos within hours of injection. This effect is never observed with saline solutions containing no or low EDT A concentrations. We have been unable to determine the specific source of the problem because the EDTA buffer is not always lethal.

Although advice to avoid EDT A may be valuable, the successful results reported by several groups using EDT A in their buffers do not support the hypothesis that EDT A is a problem. For example, Guyomard et al. (59) used a buffer containing 1 mM EDT A and observed a survival rate of 70% for rainbow trout eggs. This contrasts to results reported for goldfish by Yoon et al. (60) who report a 10% survival rate using an EDTA-free buffer. Clearly, if EDT A does affect egg viability its influence is usually small compared with other factors.

Another point about injection buffers that is worthy of comment is the estrogenic activity of phenol red (Howard Bern, personal communication). Investigators using phenol red to estimate their injection volumes may want to take this into account when evaluating their microinjection procedures. Although there appears to have been no published accounts of the effects of phenol red on developing eggs, Mart Welt (Battelle Pacific) has noted that rainbow trout eggs injected with DNA in buffer containing phenol red hatch earlier than control uninjected eggs.

Mass Gene Transfer Techniques

It is evident from the foregoing discussion that egg microinjection procedures are time consuming, laborious, species-specific and in some cases technically demanding. In addition the rate at which transgenic fish can be produced with this method is likely to be slow, particularly if genomic integration cannot be established until the FJ or F2 generations.

This slow rate of transgenic production acts as a bottleneck to the exploration, expansion and realization of the full potential of this powerful technology. Most research teams currently have a number of potentially useful constructs available for gene transfer, and it is axiomatic that with the continued rapid advancement of recombinant DNA technology there will be many more available in the near future. All of these constructs will need to be tested in order to select for the ones that best produce the desired phenotype. Research teams, working with commercial species that only breed once a year, are already faced with having to decide which construct to use. In our own case we are constrained by the shortness of the Atlantic salmon spawning season in Newfoundland and by the length of time required to handle and inject each egg. Moreover, as indicated earlier, not all eggs are of sufficient "quality" to be injected and this "quality" not only varies considerably from female to female, it also deteriorates with time following ovulation, and following artificial stripping. We discard many more eggs than are injected.


The development of non-invasive, mass transfer techniques would greatly enhance the transgenic research efforts of most groups. Not only would labor, expertise and time requirements be reduced, the high mortality rates attributable to egg handling and microinjection would be eliminated. Egg quality would not be as critical as it is now, certainly for us, and there would be no reason for this egg quality to decline following ovulation because the eggs could be used immediately.

Mass gene transfer methods would also considerably reduce the time and effort that will be required by the microinjection techniques to produce broodstock for commercial production. Aquaculture is still in its infancy; thus the best broodstock for culture and the marketplace has yet to be selected from available wild stocks. Once a gene construct appears to have significant economic value to aquaculture it would be wise to transfer it to the eggs of many different individuals in order to ensure an adequate gene pool for future breeding programs.

Potential non-invasive mass gene transfer methods include sperm binding (66), lipofection (67), particle gun bombardment (68) and electroporation (69). Our group has tried sperm binding and sperm electroporation on salmon without success to date. Daniel Chourrout reported that his group's attempts at sperm binding on rainbow trout also failed (personal communication). Tom Chen, during the early stage of his research, used the lipofection technique on sperm and found some evidence for gene transfer to rainbow trout (personal communication). However with the obvious success of the microinjection techniques most of us in the field have not investigated these mass transfer techniques as thoroughly as they perhaps should be. One of the reasons for a lack of enthusiasm in this regard is conceptual. Results obtained using microinjection techniques indicate that millions of copies of the exogenous sequence are required in order to obtain even a low frequency of integration. Therefore it is difficult to conceive how this much DNA could be transported through the micropyle by a single sperm. In addition, since many millions of sperm are required to ensure fertilization of a single egg, the proportion of sperm carrying the exogenous DNA would have to be high in order attain the integration frequencies observed using microinjection. These concerns about the potential of the mass transfer techniques account for the generally held view that they will be less efficient than microinjection, and that as a consequence, unless the new phenotype is obvious, finding the transgenic individuals may be tedious. However this supposition precludes the possibility that the spermatozoa would bring the exogenous DNA into a closer association with the fusing male and female pronuclei than microinjection.

The one mass gene transfer method that is receiving some attention recently is the electroporation of fertilized eggs. This procedure of using electric pulses to induce transient pores in the cell membrane has been successful in a number of cells including mammalian cell lines (70,71). Yamaha et al. (72) demonstrated that a number of compounds, not including DNA, could be introduced into dechorionated goldfish eggs by electroporation. Hallerman et al. (73) carried out similar experiments on fertilized dechorionated goldfish eggs in an attempt to introduce the plasmid pRSVCAT. Unfortunately they found no evidence to indicate that the DNA entered the embryo. These authors suggest that the differences between their results and those of Yamaha et al. (72) could be attributable to the lower capacitance achievable by the electroporation equipment used by Yamaha et al. (72).


The first demonstration that electroporation could be used successfully to produce transgenic fish was published by Inoue et al. (74). These authors transferred a linearized plasmid containing rainbow trout growth hormone cDNA and the mouse MIl promoter (pMV -GH) into fertilized medaka eggs with intact chorions. Twenty-five percent of the electroporated eggs hatched, of which, 4% contained the transferred sequence. Although this frequency is low relative to the 27% integration rate they obtained in a parallel study using the microinjection techniques of Ozato et al. (13), it is high enough to promote electroporation as a very viable alternative to microinjection. The potential superiority of fertilized egg electroporation over that of microinjection is implicit in the results reported by Dennis Powers at a recent workshop. He and his co-workers are finding that they can obtain integration frequencies of 25% to 75% by electroporating intact fertilized zebrafish eggs (personal communication).

FATE OF THE TRANSFERRED DNA

Replication of Injected Sequences

A number of studies have reported on the fate of injected constructs during early development. In most, if not all cases, it is evident that regardless of its form the DNA persists and is rapidly replicated during embryogenesis. Chong and Vielkind (57) clearly demonstrated, using the medaka, that supercoiled and linearized forms of the plasmid pUSVCAT were replicated and amplified 10- to 12-fold during early embryogenesis to reach peak levels at the gastrula/neurula stage. These authors also observed that the CAT DNA was amplified even when injected within intact phage particles, or as phage DNA. In a similar study with zebrafish, Stuart et al. (48) also found an approximate ten-fold increase in plasmid DNA following injection of 1, 15 or 70 pg of linearized pSV-hygro. Thus it appears that the greater the amount of DNA injected into a fertilized egg the more there is to amplify during early embryogenesis. This rapid amplification of injected DNA prior to neurulation has also been observed in the goldfish and loach (11,75). Following the gastrula/neurula stage the amount of the injected sequence present in the embryo has been shown to decline to relatively low levels in all of the fish that have been studied (goldfish, loach, zebrafish, medaka, tilapia) and it is generally associated with a smear of short DNA fragments suggestive of degeneration (11,48,56,57,75).

Conformational Changes in the Injected DNA

Injected DNA sequences undergo considerable conformational changes prior to and during their replication throughout early embryogenesis. The nature of some of these changes depends upon the initial DNA form.

Linearized Sequences

The fate of linearized sequences during embryogenesis has been followed in five fish species: goldfish (11), loach (75,76), zebrafish (48), medaka (57,64), tilapia (56). Although these studies differ considerably from one another with


respect to thoroughness and the amount of detail presented, the data are consistent enough to allow some general conclusions.

In all of the species studied the linear sequences, once injected, are converted to a high molecular weight form. This transition can take place rapidly, as evidenced by Chong and Vielkind's (57) illustration of the appearance of a high molecular weight form in zebrafish embryos within five minutes of injection. As the embryo proceeds towards gastrulation and neurulation most of the exogenous sequences become associated with the high molecular weight DNA. In addition, observations on the loach, goldfish, medaka and tilapia indicate the presence of supercoiled, open circular, closed circular and multimeric forms of the injected sequence throughout this period of embryogenesis (11,57,56,75). As the embryo completes embryogenesis, most if not all of the exogenous DNA remains in the high molecular weight form.

The nature of the high molecular weight form of the injected sequence has been examined in embryonic loach (76) and hatching medaka (57). Using genomic Southern blotting procedures the investigators concluded that their sequences were present as extended head-to-tail, head-to-head and tail-to-tail concatemers.

Supercoiled Sequences

Weare aware of four reports detailing the fate of injected supercoiled sequences, two on the medaka (57,65), one on loach (75) and one on tilapia (56).

Winkler et al. (65) examined their construct (pCMVTkCAD prior to injection, and reported that it consisted predominantly of the supercoiled and open circular forms, with a small amount of plasmid multimers. Immediately after injection into medaka embryos most of the plasmid DNA was in the relaxed form (closed circular), although some remained as supercoiled and open circular DNA. Chong and Vielkind (57) noted similar changes in their circular construct (pUSVCAT) within five minutes of injection. In both studies a high molecular weight form together with plasmid multimers (concatemers and concatenates) appeared at the blastula stage (- 6 hr), and the amounts of all DNA forms increased to a maximum by early neurulation (one day).

Following the gastrula/neurula stage the levels of all plasmid DNA forms declined. However by three days of development (30 somites) both Chong and Vielkind (57) and Winkler et al. (65) noted the persistence of supercoiled and open circular forms as well as the high molecular weight forms of the injected DNA. Winkler et al. (65) also observed closed circular and multimeric forms at this stage. The information available from tilapia suggests that circular sequences undergo similar conformational changes to those observed in the medaka. Phillips (56) examined three-day embryos (one day before hatch) and found evidence for supercoiled, linear, open circular as well as high molecular weight forms.

Chong and Vielkind (57) only found evidence for the high molecular weight form of the plasmid DNA at hatching of the eggs, suggesting that all of the other forms had been degraded. The same result was obtained by Phillips (56) when he examined nine-day-old tilapia fry. However, Winkler et al. (65) demonstrated, in the medaka, that various forms of plasmid DNA can persist for long periods of time after hatch (see later discussion).

Chong and Vielkind (57) examined the nature of the high molecular weight form in medaka at the time of hatching. They concluded from genomic Southern analysis that the injected supercoiled plasmids had been rearranged into multimeric


circles or long tandem arrays of identical head-to-taillinear monomers. There was no evidence for the head-to-head or tail-to-tailligations observed when the linear plasmid was injected into the eggs. In contrast, Kozlov et al. 's (76) restriction analysis did not reveal evidence of intact multimers in the 9 hr loach following injection of the plasmid AIl53. Instead, their results indicated rearrangement of the restriction sites within the plasmid.

Persistence of Extrachromosomal DNA in Juveniles and Adults

Most ofthe foregoing results and discussion are derived from data obtained from tropical or warm-water species where embryological development is relatively rapid. How much of it applies to the slower developing cold-water species, such as salmonids, remains to be determined. For the present it seems reasonable to assume that the sequence of changes the exogenous DNA undergoes following injection does not differ greatly between species. Species differences are likely at the quantitative level because of differences in the rates of extrachromosomal DNA synthesis and degradation. We do know that nuclease activity can differ widely between cell types and fish species.

The working hypothesis that emerges from the foregoing studies on warmwater and tropical species is that most of the DNA injected into fertilized eggs persists extrachromosomally where it is chemically modified and replicated during embryogenesis. Most authors are of the opinion that the increasing levels of injected sequences during early embryogenesis is an indirect result of the rapid rate of cell division, and thus chromosomal duplication, taking place during this time. At this point the rate of extrachromosomal DNA synthesis appears to exceed its rate of degradation. Once neurulation has been completed the levels of extrachromosomal DNA decline, suggesting that degradation rates have begun to exceed synthesis. Kozlov et al. (76), referring to data generated from experiments with sea urchins (81,82), suggested that the amplification of foreign DNA during early embryogenesis is related to the largely cytoplasmic distribution of DNA polymerases, and the decline in the levels of these exogenous sequences occurs when the polymerase migrates into the embryonic nucleus. Regardless of the validity of this hypothesis, it does focus on the possibility that controls over DNA replication may be relatively loose during the initial stages of embryogenesis, and that these controls tighten as the embryo develops.

One consequence of the extrachromosomal nature of the exogenous sequences is that they cannot be distributed equally between daughter cells. This fact, along with the possibility that high levels of extrachromosomal sequences can only be maintained in rapidly-dividing cells, results in the production of mosaic tissues, organs and individuals. The mosaic pattern of injected DNA (chicken 8-crystallin gene) persistence and expression in embryonic medaka, is a clear indication of how unequal the distribution between cells can be (13,77).

The persistence of large amounts of extrachromosomal DNA complicates our ability to determine whether or not genomic integration of any of the exogenous sequences has occurred. The fact that the injected DNA can be modified and rearranged adds to the problem. Genomic Southern procedures may not allow the distinction between junction fragments with recipient DNA, and rearrangement of restriction sites within the plasmid.

The picture that emerges from studies of the tropical fish species is that the level of injected DNA in the embryo declines following the gastrula/neurula stage.


If the results obtained by Stuart et al. (48) can be generalized, it would appear that the length of time this largely extrachromosomal DNA persists will depend upon the amount injected and amplified during early embryogenesis. In other words, the more extrachromosomal DNA present, the longer it will persist. Since the fish are mosaic for the exogenous DNA it is evident that this hypothesis also predicts that some cells will retain their extrachromosomal DNA longer than others.

How long can extrachromosomal DNA persist within the fish? In the two species that have been examined at hatch (zebrafish, medaka), all of the individual fish possess the exogenous sequences. However from this point onward the proportion of fish containing these sequences declines. Chong and Vielkind (57) noted that four weeks after hatch the number of injected medaka retaining foreign sequences (injected as linear or supercoiled molecules) had declined to 20 to 30%. All of the exogenous DNA was in a high molecular weight form. Winkler et al. (65) found that a similar proportion (25%) of their medaka injected with supercoiled DNA contained the exogenous sequence at three months of age. However in this case most of the exogenous sequences persisted as extrachromosomal supercoiled and open circular DNA. There were only traces of the high molecular weight form present. By the time Winkler et al.'s (65) medaka had reached nine months of age the percentage of fish retaining the exogenous sequence had declined to 5%. In the one fish that was analyzed, only the high molecular weight form remained. Stuart et al. (48) in their study of the zebrafish reported that although most of their three-week-old fish showed evidence of retaining the exogenous linear sequence, it was present on average at less than one copy per cell; by the time they were four months old 95% of them had lost the sequence. Kozlov et al. (76) noted that while most of their 8-day loach embryos showed evidence for the presence of the exogenous plasmid, only 2 out of 42 (5%) retained the sequence after 30 days of development.

The decline with growth and age in the proportion of fish containing exogenous genes is highly suggestive that all of the injected DNA is extrachromosomal in most embryonic and hatching fish. In addition, although incomplete, the data also suggest that the decline in the number of fish carrying exogenous sequences, whether injected in the linear or supercolied form, can be relatively slow. Furthermore, if the observations of Stuart et al. (48) on embryonic zebrafish can be extrapolated to post-hatched juveniles, the length of time required to clear the extrachromosomal DNA from the fish may be positively related to the amount of DNA injected into the fertilized eggs. The point of concern here is that extrachromosomal DNA may persist in some fish until sexual maturity. The presence of supercoiled and open circular forms in three-month-old medaka is clear evidence of this phenomenon when supercoiled sequences are injected (65).

There is a paucity of data on the fate of the injected DNA during development and growth of colder-water species. However the available information appears to be consistent with observations made on the warm-water or tropical fish. The following is a summary of some of the results pertaining to salmonids.

Rokkones et al.'s (42) data on hGH mRNA production by rainbow trout embryos (pre-hatch) suggest at least 72% of them contained the injected plasmids (MThGH). Chourrout et al. (53) examined rainbow trout embryos at hatch and found that 40% retained the injected (200 pg) supercoiled sequence, while 75% retained the same sequence injected in the linear form. McEvoy et al. (78) injected Atlantic salmon eggs and found evidence to suggest that after fourteen weeks of


development (alevins) approximately 50% of them retained the foreign DNA. In a more complete study, Guyomard et al. (59) found that 40% of their 6- to 12-month-old rainbow trout retained the exogenous DNA when 200 pg of linearized sequences were injected into fertilized eggs. When 500 pg of a similar linear sequence were injected, 74% of them retained the exogenous DNA for at least six months. Southern blot analysis of the latter group of fish revealed evidence suggesting that a significant portion of the foreign DNA was present as large extrachromosomal concatenates. Rokkones et al. (42) injected large amounts of DNA (5000 pg) and found that 75% of their 73-day-old rainbow trout alevins contained either the injected supercoiled or linearized construct, while one year after injection one out of five (20%) Atlantic salmon retained evidence of the injected supercoiled plasmid. Penman et al. (58) injected relatively small amounts of DNA (7 pg) and found on average that only 6.9% and 6.8% of the late alevins and early fry, respectively, retained evidence of the linearized sequence. They also found that approximately 4% of the early fry retained the supercoiled sequence. Furthermore genomic Southern analyses revealed that in most of these fish the bulk of the supercoiled sequences remained as extrachromosomal multimers. In our work on Atlantic salmon we normally inject small amounts of DNA (~ 12 pg) and have never found foreign DNA retention rates exceeding 6%. Our long-term average following the injection of thousands of eggs is approximately 2%.

A close examination of all of the data presented for salmonids injected with 200 or more pg of DNA reveals a significant (P < 0.05) negative correlation between the proportion of fish carrying exogenous sequences and age. Thus it appears that salmonids, in keeping with their tropical counterparts, may gradually lose exogenously-administered DNA over time.

It is evident from the results obtained from tropical and cold-water fish species that foreign DNA, whether injected as the linear or supercoiled form, can persist extrachromosomally for long periods of time, and may be present in the fish when it is reproductively active. If it is present in the germ cells, we are aware of no reason why it cannot be passed on to the next generation, such as has been reported for the mouse and nematode (C eJegans) (79,80).

Another, more hypothetical, conclusion that can be drawn from the data on fish is that the probability of extrachromosomal DNA persisting until sexual maturity increases with the amount of DNA injected into the eggs: the "DNA load-dependent extrachromosomal' hypothesis. This hypothesis is founded on four main observations. I) Most of the injected DNA remains extrachromosomal during early embryogenesis (11,56,57,65,75). 2) The length of time required to clear extrachromosomal DNA from zebrafish embryos is directly related to the amount injected into the eggs (48). 3) The proportion of fish containing exogenous sequences declines relatively slowly as the free-swimming fish grow and age (48,65, see also above discussion on salmonids). 4) Extrachromosomal DNA can persist until the fish are sexually mature (59,65).

In an earlier section of this review we assembled the available data to suggest that the potential to produce transgenic salmonids increases considerably as the amount of DNA injected into the eggs is increased: "DNA load-dependent integratiod' hypothesis (Table 5). The question provoked by the "DNA loaddependent extrachromosomal' hypothesis is how much of the observed increase in fish carrying the exogenous sequence is entirely attributable to the presence of extrachromosomal DNA.


Given the current difficulties encompassed in demonstrating chromosomal integration it will be difficult critically to evaluate the above two hypotheses. In addition there is no evidence to indicate that they are mutually exclusive. Thus in order to obtain a high frequency of germline transformants it may be necessary to accept the presence of extrachromosomal DNA. However if this is the case it could take a number of generations to select stable lines of transgenic fish, a difficult proposition when the generation times of most commercial species is taken into account.

Genomic Integration

The present state of our ignorance concerning the factors that are important to successful genomic integration makes it difficult to make useful comment on this subject.

Most investigators now carry out restriction analyses and genomic Southern blotting procedures in attempts to establish whether the introduced DNA is covalently integrated into the genome. The criteria used to argue for an integration event include co-migration with the host high molecular weight DNA when undigested, and, with the use of appropriate restriction endonucleases, demonstration of the presence of one or more copies of the intact gene, and the presence of restriction fragments suggestive of a covalent linkage between this gene and the genomic DNA.

The general conclusion that can be derived from the published Southern blot evidence is that, when present, the foreign gene persists intact as concatemers of varying length that can be orientated in all of the expected configurations: head-to-head, head-to-tail and tail-to-tail. Evidence for junction fragments of various sizes has been presented in most cases. However, as indicated earlier, all investigators recognize that fragments larger than those predicted from the restriction map of the injected sequence could also represent rearrangements of the foreign DNA following injection. Clearly, as stated by Chourrout et al. (5), it is much easier to demonstrate the presence of extrachromosomal DNA, using restriction analyses, than it is to prove integration. Restriction fragments representing the free ends of the injected sequence have been found in a number of cases (57-59). Unfortunately, evidence for the presence of extrachromosomal DNA does not preclude the possibility that an integration event has taken place.

There are difficulties encompassed in establishing irrefutable evidence of genomic integration in the Fo' Although the cloning and sequencing of putative junction fragments could provide more convincing evidence, it is clearly not feasible to do this routinely at the present time. Even if it were possible, the mosaic nature of most transgenic fish to date would necessitate an examination of the germ cells themselves, rather than the more readily available blood cells or fin clips.

INHERITANCE

There are now numerous examples of the passage of foreign DNA to the next generation (F1) in species such as medaka (74), trout (59), zebrafish (48,63) and carp (32). Mosaicism in the parents leads to a wide variation in the percentage of offspring carrying the foreign DNA. This is illustrated in the study by Zhang


et al. (32) where the passage of an RSV - rainbow trout growth hormone cDNA construct was followed into the FI.-..generation of carp. Four Fo males that were positive for the presence of the cuNA in fin tissue were crossed to the same female. Progeny from the four crosses showed 32.3%, 42.3%, 100% (based on only four individuals) and 0% transmission, respectively. The latter result suggests that the foreign DNA was not integrated into the germ line of this male parent. A similar finding is reported in zebrafish; Stuart et al. (63) report that eight out of ten fish that expressed an RSV /SV40 CAT construct failed to pass on the gene to the FI generation. Conversely, two fish which had failed to express CAT activity in fin tissue proved to be germline transformants.

The well-documented pattern of amplification and persistence of injected foreign DNA as extrachromosomal elements, together with reports of the passage of extrachromosomal DNA to the next generation in both mouse (79) and C eJegans (80), has led to caution in interpreting the presence of the DNA in FI individuals. In the absence of direct proof of stable inheritance through integration, researchers have relied on two sources of evidence.

The first is based on the reproducible pattern of expression of the transgene in the F). generation. Stuart et al. (63) report that CAT activity in F l individuals derived rrom the RSV /S V 40 CAT construct microinjection series is umformly well expressed in fin and skin, with lower levels found in heart, gill and muscle and little or no CAT activity seen in brain, liver and gonad. This pattern of expression is consistent with inheritance through integration, with tissue specificity being directed by the viral promoter/enhancer combination. However, the persistence of extrachromosomal DNA into the next generation is more likely to lead to further mosaicisms in the pattern of CAT activity in different tissues.

The second line of evidence is that the distribution of the transgene in the F 2 generation is consistent with Mendelian inheritance. In 1988, Stuart et al. (48) reported on the passage of a recombinant plasmid, pSV-hygro, through Fo and FI generations to the F 1. progeny. Of the F 0 individuals surviving from microinjection, 5% of the fish retamed the DNA in their fins after four months. One of these individuals passed on the foreign gene to 20% of her offspring, consistent with mosaicism. However, when the F2, progeny were analyzed, the transmission rate was 50%, as expected from a single, heterozygous genomic insert. A subsequent study (63) with pUSV CAT as the foreign DNA injected in zebrafish independently confirmed these findings. When F f progeny derived from four transgenic F 0 fish were tested for inheritance of the CAT gene, 53%, 48%, 56% and 53% proved to be positive. Their parents came from cohorts that were respectively 36%, 54%, 12% and 25% positive for the transgene. Again, these results are indicative of the inheritance of a single, heterozygous genomic insert. In contrast, Inoue et al. (74), studying transmission in medaka of a recombinant plasmid pMV -GH electroporated into fertilized eggs, reported that 100% of the F I progeny derived from the cross of a transgenic male and wild-type female possessed the transgene and that the gene was present in 88% of the F l progeny. The authors suggest that independent integration of the transgene mto both sets of chromosomes could account for the high percentage of transmission.

When inheritance analyses must stretch to the F generation and beyond, there are obvious advantages to using zebrafish and meJaka. This emphasizes the need for such model systems to be used in conjunction with any applied aquaculture projects. Also, with the need to wait until the F I and F 2 generations before confirming inheritance, there is a real need for facile screening procedures


based on the polymerase chain reaction (PCR) to detect the presence of the transgene and eventually the sex of the fish. We have used PCR to detect antifreeze protein genes in minute quantities of blood and milt of F 0 salmon in order to select fish for breeding. This method also has enabled us to screen Fl progeny as soon as they can be tagged and bled. In addition to the statistica information generated, there is the practical advantage of being able to conserve tank space by discarding non-transgenic F) and F2 individuals.

GENE EXPRESSION IN TRANSGENIC FISH

There are two components to the transgenes transferred to fish, the regulatory region made up of a promoter/enhancer combination and the structural portion of the gene. Although they cannot be considered completely independently of each other, "use any combination that works" seems to be the maxim in this developmental stage of the technology. Thus the example of a mammalian promoter driving expression of a bacterial gene in a fish (78) is typical of the "cut and paste" constructs that are currently in use, although there are exceptions. The winter flounder antifreeze protein gene and its promoter were transferred intact and expressed in salmon (29), and the expression of the chicken delta-crystallin gene directed by its native promoter has been studied in medaka during embryogenesis (77). Typically, researchers have opted to use the most powerful promoters that have activity in a wide range of tissues. These include viral promoters like RSV, SV40 and Mouse Mammary Tumor Virus (MMTV) and the strongly inducible metallothionein promoter, that has served so well in the transgenic mouse (3). For the selection of the structural gene portion there have been two or three options. Researchers have either used reporter genes such as CAT (57,63,97), beta-galactosidase (78) and delta-crystallin (77) as an efficient way of studying expression of the transgene, or they have chosen genes of commercial interest such as growth hormone (32,42,55,59,74), or antifreeze (29). In this latter category one could include genes for antibiotic resistance (48,60) that would lead to a method for the direct selection of transgenic individuals.

Regulatory Regions

The success of driving transgene expression with foreign enhancer/ promoters stems from the conservation of their ds-actipg regulatory sequences which allows them to be recognized by host Inlns-acting factors. These bipartite protein factors have a site for recognizing and binding the ds-acting DNA sequence and a more amorphous region responsible for contacting the transcritr tional apparatus (98,99). Although recognition and binding of the DNA sequence is crucial, enhancer/promoters are usually a composite of several individual binding sites such that their transcriptional efficiency comes from the summation of multiple interactions. Thus, the failure of one cis-acting sequence to be recognized might reduce but not eliminate the action of an enhancer/promoter. The efficacy of these regulatory regions in a transgenic host is determined empirically with the extrapolation from other systems. One useful proving ground for gene constructs has been tissue culture. A variety of fish cell lines have been used for the transient expression of gene chimeras that usually incorporate a reporter gene or a gene with a product that is easily assayed (100,10 I). F riedenreich and Schartl


have assayed many pennutations of promoters, enhancers and structural genes in several cell lines (l 0 1). In general, the enhancer/promoter combinations that function well in tissue culture are active in the transgenic host.

The two most popular regulatory sequences are the mouse metallothionein (MT-I) and RSV enhancer/promoters. The MT-I enhancer/promoter is constitutively active in a variety of tissues, notably kidney, liver and intestine (l02). Its activity can be increased by administration of certain transition or heavy metal ions, although this has little relevance to practical applications in aquaculture. The MT -1 promoter has been linked to growth honnone genes for injection into a variety of fish, carp, catfish, goldfish, loach, medaka, salmon and trout (ref. 3; Table 3), and has driven expression in some of these systems. There are examples where the transgenic fish and their offspring have a significantly greater mean weight than the uninjected control fish. Maclean et al. (75) report success with transgenic loach that received the mouse MT -1 promoter coupled to the human growth honnone gene. So too, do Chen et al. (61) for silver crucian carp that were microinjected with the same gene chimera.

The RSV enhancer/promoter, derived from the long terminal repeat section of the avian Rous sarcoma virus, can direct gene expression in most, if not all, tissues but is particularly active in those of mesodennal origin such as muscle and skin. The usefulness of this tissue preference has been commented on above where the reproducible pattern of RSV-CAT expression in tissues of Fl progeny of transgenic zebrafish was good evidence of integration (63). However, its basal activity in a wide range of tissues was the reason it was chosen by Y oon et al. to drive expression of the bacterial neomycin phosphotransferase (neo) gene in transgenic goldfish (60). The objective here was to select for transgenic hatchlings on the basis of their resistance to the antibiotic G418, a neomycin analog. Although the results were inconclusive in F 0 transgenic fish, probably because of mosaicism, the experiment should be repeated with the F 1 generation. A practical success has come from coupling this promoter to a truncated rainbow trout growth honnone cDNA for microinjection into carp (32). Even though the DNA sequence coding for the growth honnone signal polypeptide was missing from the construct, transgenic F Jish expressed the honnone and showed increased growth rates over control fish. 1 his phenotype was also observed for the F 1 generation derived from crosses of these fish.

Refinement of the Regulatory Sequences

One predictable problem in the application of these control sequences in aquaculture is potential resistance of consumers and regulatory agencies to the use of sequences that are foreign to fish. It is not clear at this time what degree of chimerism or evolutionary distance will be acceptable for transgenes in fish. Although it is difficult to guess how this issue will be perceived by the public in years to come, one might anticipate problems from the use of a mouse DNA or sequence from an avian retrovirus. At the same time there will be need for more varied and sophisticated strategies for transgene expression, as discussed below. The combination of these two needs will require the isolation of a variety of fish genes and the characterization of their enhancer/promoter sequences. Despite good arguments for establishing fish as model vertebrates, not a great deal of curiositymotivated research is funded in this area. This is evident from the current paucity of fish gene sequences in the database. Byrnes and Gannon (103) have responded


to this need by cloning a battery of cDNAs expressed in the liver of Atlantic salmon. Among the clones they isolated was the cDNA for serum albumin which appeared to be the most abundant sequence in the liver library.

Structural Genes

The importance of reporter genes in model studies has been mentioned previously. The bacterial CAT gene is particularly useful because its expression can be detected at extremely low levels. The CAT activity is easier to detect and quantitate than its DNA sequence. Indeed there are examples where CAT activity has been measured, but the CAT gene cannot be detected by hybridization (97). There will be a continuing need for this and other reporter genes in testing new enhancer/promoters in tissue culture and the whole animal.

The popularity of growth hormone expression, particularly in targeted research programs, is obvious. It promises improved feed conversion and a decreased cultivation time before harvesting. These are both very important considerations economically in such a competitive business. The initial successes with this gene, where the rate of growth or mean average size of fish expressing elevated levels of growth hormone have increased (61), are very encouraging. However, it is still too early to say if the side effects to increased growth hormone production that have plagued transgenic livestock will surface in fish. Even so, this problem with domestic animals probably stems from the inappropriate regulation or overexpression of the growth hormone transgene rather than a flaw in the overall concept. As more sophisticated regulatory schemes are introduced problems with side effects will be minimized.

In contrast, the problems in obtaining appropriate antifreeze protein expression in transgenic fish are just the opposite. Unlike growth hormone, antifreeze protein must be produced in vast quantities if it is to serve its function of conferring protection on the host. Natural producers have circulating levels of 5 to 25 mg/ml of serum (9) and invariably this concentration of antifreeze protein is produced by multiple gene copies (35). This may be a reflection of the natural promoters not being very strong. In initial attempts to express antifreeze protein in Atlantic salmon, one of the tandem repeats from the antifreeze protein gene clusters (104) in winter flounder was microinjected into salmon (50). Because it was a complete tandem repeat all of the control sequences for transcription should have been present. The level of antifreeze protein expression obtained from one or several copies of the transgene has predictably been sufficient for detection immunologically but not by freezing point depression. If there was formation and integration of a head-to-tail concatemer of the repeat as reported in many other transgenic systems, not only would the gene dosage increase but the structure of the resulting gene cluster would essentially be the same as that found in the winter flounder. An alternative strategy to overproduce the antifreeze protein might be to switch over to a more powerful liver-specific promoter.

Future choices for structural genes will almost certainly include genes for disease resistance. As the technology develops, antisense and ribozyme constructs designed to attenuate or eliminate viral messenger RNAs will become increasingly feasible.

There are other facets to the expression of a transgene in a foreign host that are beyond the scope of this review. Two issues are worth mentioning, however. One is the role of introns in a stimulating type expression of a transgene.


In this field of research cDNA and genomic genes have been used almost interchangeably, and yet there are clear indications that the presence ofintrons can be very beneficial to expression in some systems (105). The other issue which is beginning to emerge is the value of matrix association regions (MAR) in protecting a transgene from position effects (106). It might well be advisable to incorporate MARs flanking the introduced transgene to protect it from domination by neighboring genetic elements.

TRANSGENIC FISH FOR AQUACULTURE: POTENTIAL BONANZA OR "RED HERRING"?

There are many improvements in the genetic makeup of commercial fish that we would like to make using transgenic technology. However, eight years after Brinster and colleagues' dramatic demonstration that mice could be rendered giants by the addition of a few extra growth hormone genes, it is fair to say that the initial optimism about the rapid production of economically useful transgenic fish has given way to the more rational realization that progress will be gradual. It is clear that we cannot, in the near future, fulfill all dreams of genetically superior broodstock. Thus we must focus on realistic goals, keeping in mind environmental concerns as well as the fact that the growers are producing food for human consumption.

Given our present knowledge of gene regulation, particularly in fish, it is only possible to target traits controlled by single genes. In isolating and cloning these genes, it is our opinion that emphasis should be placed on fish genes, for it is likely that gene constructs from other organisms (human, bovine, mice, viruses) will be I:ejected at the marketplace.

At this point in time very few phenotypically useful fish genes are available, the most notable being growth hormone and antifreeze proteins. The genes selected for transfer must be expressed at levels appropriate for their physiological function. In addition, it may also be necessary to have expression take place at specific developmental stages and/or within specific tissues. This will require considerable research into the molecular physiology of fish in order to identify and isolate appropriate promoters, enhancers, silencers and tissue-specific elements. From the practical standpoint, exogenous control over transferred genes in commercial species cannot be done on an individual basis. If regulation of expression is necessary, it must be done without handling the fish. This could be accomplished by manipulating diet, photoperiod, temperature, salinity or, more futuristically, by utilizing responses elicited by the fish,es' chemosensory system. Clearly since almost nothing is known about how these factors regulate genes in fish we have exciting prospects for future research.

At present, the fish that are most likely to benefit from the transgenic technology are those in which culture techniques have already been well defined (e.g., salmonids, catfish), However, because of their large size, short reproductive season, slow growth rates and long generation times these fish are not the best models in which to test expression and heritability of gene constructs. In forthcoming years, researchers will take greater advantage of fish cell lines and model fish (e.g., tilapia, zebrafish) with short generation times, to screen gene constructs for integration, expression and heritability.


There are two essential elements to the successful use of transgenic fish for food. The end product must be safe for the environment as well as for human consumption, and the end product must be believed to be safe by the public at large. Let us hope our "red herring" is a "straw man". However if transgenics are to be a bonanza to food production we must be as innovative and honest in public relations as we are in science.

Acknowledgments: We would like to express our appreciation to all of the investigators who helped enormously in the writing of this review by providing us with unpublished manuscripts, recent results and advice: Daniel Chourrout, Tom Chen, Rex Dunham, Frank Gannon, Steve Goddard, Patrick Gibbs, Kevin Guise, Norman Maclean, Kenjiro Ozato, Peter Phillips, Dennis Powers, Erik Rokkones, Allan Vaisius, Juergen Vielkind, Marc Welt and Zuo Van Zhu. We are also grateful for our colleagues Choy Hew and David Idler and members of the research team, Madonna King, Sherry G~uthier and Sally Goddard who make our contribution to this field possible. We are indebted to Dr. Margaret Shears for producing all of our transgenic salmon, particularly when she, on two separate occasions, was at the point of giving birth to her own children. The research of P.L.D. and G.L.F. is supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada.

REFERENCES

1 Hew, C.L. (1989) Fish Physio1. Biochem. 7, 409-413. 2 Ozato, K., Inoue, K. and Wakamatsu, Y. (1989) Zoo1. Sci. 6,445-457. 3 Chen, T.T. and Powers, D.A. (1990) Tibtech. 8,209-216. 4 Powell, R., Byrnes, L. and Gannon, F. (1990) Genetic Engineer and

Biotechnologist 10, 7-9. 5 Chourrout, D., Guyomard, R. and Houdebine, L.M. (1990) in Transgenic

Models in Medicine and Agriculture (Church, R.B., ed.) pp. Wiley-Liss, Toronto, pp. 89-99.

6 Fletcher, G.L., Kao, M.H. and Dempson, J.B. (1988) Aquaculture 71, 369-378.

7 Saunders, R.L. (1986) Inst. Freshwater Res. Drottningholm Rep. No. 63. 8 Aiken, D.E. (1986) Can. Aquacult, 2, 27-29. . 9 Davies, P.L., Hew, C.L. and Fletcher, G.L. (1988) Can. J. Zoo1. 6,2611-

2617. 10 Hallerman, E.M. and Kapusinski, A.R. (1990) Fisheries 15, 12-20. 11 Zhu, Z., Li, G., He, L. and Chen, S. (1985) J. App1. Ichthyo1. 1, 31-34. 12 Rokkones, E., Alestnj>m, P., Skjervold, H. and Gautvik, K.M. (1985) Acta

Physio1. Scand. 124, Suppl 542, 417. 13 Ozato, K., Kondoh, H., Inohara, H., Iwamatsu, T., Wakamatsu, Y. and

Okada, T.S. (1986) Cell. Differ. 19,237-244. 14 Markert, J.R., Higgs, D.A., Dye, H.M. and MacQuarrie, D.W. (1977) Can.

J. Zoo1. 55, 74-83. 15 Higgs, D.A., Donaldson, E.M., McBride, J.R. and Dye, H.M. (1978) Can.

J. Zoo1. 56, 1226-1231. 16 Sekine, S., Mizukami, T., Nishi, T., Kuwana, Y., Saito, A., Sato, M., Itoh,

S. and Kawauchi, H. (1985) Proc. Nat. Acad. Sci. U.S.A. 82, 4306-4310.


17 Gill, 1.A., Sumpter, 1.P., Donaldson, E.M., Dye, H.M., Souza, L., Berg, T., Wypych, 1. and Langley, K. (1985) Bioffechnology 3, 643-646.

18 Wagner, G.F., Fargher, R.e., Brown, 1.e. and McKeown, B.A. (1985) Gen. Compo Endocrinol. 60, 27-34.

19 Kawauchi, H., Moriyama, S., Yasuda, A., Yamaguchi, K., Shirahata, K., Kubota, 1. and Hirano, T. (1986) Arch. Biochem. Biophys. 244, 542-552.

20 Weatherly, A.H. and Gill, H.S. (1987) Aquaculture 65, 55-66. 21 Agellon, L.B., Emery, CJ., Jones, 1.M., Davies, S.L., Dingle, A.D. and

Chen, T.T. (1988) Can. 1. Fish. Aquat. Sci. 45, 146-151. 22 Down, N.E., Donaldson, E.M., Dye, H.M., Langley, K. and Souza, L.M.

(1988) Aquaculture 68, 141-155. 23 Down, N.E., Donaldson, E.M., Dye, H.M., Boone, T.e., Langley, K.E. and

Souza, L.M. (1989) Can. 1. Fish. Aquat. Sci. 46, 178-183. 24 Danzmann, R.G., Van Der Kraak, GJ., Chen, T.T. and Powers, D.A.

(1990) Can. 1. Fish. Aquat. Sci. 47, 1292-1301. 25 Komourdjian, M.P., Burton, M.P. and Idler, D.R. (1978) Gen. Compo

Endocrinol. 27, 240-253. 26 Fletcher, G.L., Kao, M.H. and Fourney, R.M. (1986) Can. 1. Zool. 64,

1897-1901. 27 Cutler, AJ., Saleem, M., Kendall, E., Gusta, L.V., Georges, F. and

Fletcher, G.L. (1989) l Plant Physiol. 135, 351-354. 28 Kao, M.H., Fletcher, G.L., Wang, N.C. and Hew, e.L. (1986) Can. 1. Zool.

64, 578-582. 29 Davies, P.L., Hew, e.L., Shears, M.A. and Fletcher, G.L. (1990) in

Transgenic Models in Medicine and Agriculture (Church, R.B., ed.) pp. 141-161, Wiley-Liss, Toronto.

30 Du, SJ., Gong, Z.Y., Fletcher, G.L., Shears, M.A. and Hew, C.L. (1990) in Transgenic Fish (Hew, e.L. and Fletcher, G.L. eds.) World Scientific Publications, Singapore.

31 Zhu, Z., Xu, K., Li, G., Xie, Y. and He, L. (1986) Kexue Tongbao 31, 988-990.

32 Zhang, P., Hayat, M., Joyce, C., Gonzalez-Villasenor, L.I., Lin, e.M., Dunham, R.A., Chen, T.T. and Powers, D.A. (1990) Mol. Reprod. Dev. 25, 3-13.

33 Guise, K.S., Hackett, P.B. and Faras, AJ. (1990) in Transgenic Fish (Hew, e.L. and Fletcher, G.L. eds.) World Scientific Publications, Singapore.

34 Fletcher, G.L., Idler, D.R., Vaisius, A. and Hew, e.L. (1989) Fish. Physiol. Biochem. 7, 387-393.

35 Davies, P.L., Fletcher, G.L. and Hew, e.L. (1989) in Oxford Surveys of Eukaryotic Genes (Maclean, N., ed.) 6, 85-109, Oxford University Press, Oxford.

36 Powers, D.A. (1990) Science 246, 352-358. 37 Barinaga, M. (1990) Science 250, 34-35. 38 Nash, C.E. and Kensler, e.B. (1990) World Aquaculture 21, 104-112. 39 Ginsburg, A.S. (1968) Fertilization in fishes and the problem of polysper

my. Academy of Sciences of the USSR, Institute of Developmental Biology. Translated from Russian by Z. Blake and edited by B. Golek. Israel Program for Scientific Translations, Jerusalem 1972, Keter Press Binding: Wiener Binding Ltd, Jerusalem, 366 p.

40 Brummet, A.R. and Dumont, IN. (1979) l Exp. Zool. 210, 417-433.


41 Strojek, R.M. and Wagner, T.E. (1988) in Genetic Engineering (Set1ow, J., ed.) 10,221-246, Plenum Press, New York, NY.

42 Rokkones, E., Alestrom, P., Skjervold, H. and Gautvik, K.M. (1989) J. Compo Physiol. 158, 751-758.

43 Hammer, R.E., Pursel, V.G., Rexroad, C.E., Jr., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D. and Brinster, R.L. (1985) Nature (London) 315, 680-683.

44 Yamaha, E., Onozato, H. and Yamazaki, F. (1988) Nippon Suisan Gakkaishi 54, 537.

45 Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. and Palmiter, R.D. (1985) Proc. Nat. Acad. Sci. U.S.A. 82, 4438-4442.

46 Dunham, R.A., Eash, J., Askins, J. and Townes, T.M. (1987) Trans. Amer. Fish. Soc. 116, 87-91.

47 Korzh, V.P. (1982) Soviet J. Develop. BioI. 12, 134-137. 48 Stuart, G.W., McMurray, J.V. and Westerfield, M. (1988) Development

103, 403-412. 49 Hallerrnan, E.M., Schneider, J.F., Gross, M.L., Faras, A.J., Hackett, P.B.,

Guise, K.S. and Kapuscinski, A.R. (1988) Trans. Amer. Fish. Soc. 117, 456-460.

50 Fletcher, G.L., Shears, M.A., King, M.J., Davies, P.L. and Hew, C.L. (1988) Can. J. Fish. Aquat. Sci. 45, 352-357.

51 Fletcher, G.L., Shears, M.A., King, M.J., Kao, M.H., Davies, P.L. and Hew, c.L. (1990) in Fish Physiology, Fish Toxicology and Fisheries Management: Proceedings of an International Symposium, Guangzhou, PRC, September 14-16, 1988 (Ryans, R.C., ed.) United States EPA Environmental Research Laboratory, Athens, GA. EP A/600/9-90/0 11, pp. 230-247.

52 Maclean, N., Penman, D. and Talwar, S. (1987) Proc. World Symp. on Selection, Hybridization and Genetic Engineering in Aquaculture. Bordeaux, May 27-30, 1986, Vol. II, pp. 325-333, Berlin. .

53 Chourrout, D., Guyomard, R. and Houdebine, L.M. (1986) Aquaculture 51, 143-150.

54 Gibbs, P.D.L., Gray, A. and Thorgaard, G.H. (1988) Congress abstracts Aquaculture International Congress and Exposition. Vancouver Trade and Convention Centre, Vancouver, B.C., Canada, Sept. 6-9, 1988, p. 56.

55 Brem, G., Brenig, B., Horstgen-Schwark, G. and Winnacker, E.L. (1988) Aquaculture 68, 209-219.

56 Phillips, P .C. (1989) In vitro fertilization and gene transfer in tilapia. Ph.D. dissertation, Dept. Zoology, Southern Illinois University at Carbondale.

57 Chong, S.S.C. and Vielkind, J.R. (1989) Theor. Appl. Genet. 78, 369-380. 58 Penman, D.J., Beeching, A.J., Penn, S. and Maclean, N. (1990) Aquacul

ture 85, 35-50. 59 Guyomard, R., Chourrout, D., Leraux, C., Houdebine, L.M. and Pourrain,

F. (1989) Biochemie 71, 857-863. 60 Yoon, S.J., Hallerrnan, E.M., Gross, M.L., Liu, Z., Schneider, J.F., Faras,

AJ., Hackett, P.B., Kapuscinski, A.R. and Guise, K.S. (1990) Aquaculture 85,21-33.

61 Chen, T.T., Lin, C.M., Zhu, Z., Gonzalez-Villasenor, L.I., Dunham, R.A. and Powers, D.A. (1990) in Transgenic Models in Medicine and Agriculture (Church, R.B., ed.) pp. 127-139, Wiley-Liss, Toronto.


62 Powers, D.A., Gonzalez-Villasenor, L.I., Zhang, P., Chen, T.T. and Dunham, R.A. (1990) Proc. of Conference on Transgenic Technology in Medicine and Agriculture (1989) NIH (in press).

63 Stuart, G.W., Vielkind, l.R., McMurray, J.V. and Westerfield, M. (1990) Development 109, 577-584.

64 Vielkind, J.R., Chong, S.S.S., Stuart, G.W. and Sadaghiani, B. New Trends in Ichthyology (Schroeder, J.H., ed.) Paul Parey Publisher, Germany (in press).

65 Winkler, c., Vielkind, J.R. and Schartl, M. Mol. Gen. Genet. (in press). 66 Lavitrano, M., Camaioni, A., Fazio, V.M., Doici, S., Farace, M.G. and

Spadafora, C. (1989) Cell 57, 717-723. 67 FeIgner, P.L., Gadek, T.R., Holm, R., Chan, H.W., Wenz, M., Northrop,

J.P., Ringold, G.M. and Danielsen, M. (1987) Proc. Nat. Acad. Sci. U.S.A. 84,7413-7417.

68 Klein, T.M., Wolf, E.D., Wu, R. and Sanford, J.c. (1987) Nature 327, 70-73.

69 Shigekawa, K. and Dower, WJ. (1988) BioTechniques 6, 742-751. 70 Neumann, E., Schaefer-Ridder, M., Wang, Y. and Hofschneider, P.H.

(1982) EMBO J. 1,841-845. 71 Potter, H., Weir, L. and Leder, P. (1984) Proc. Nat. Acad. Sci. U.S.A. 81,

7161-7165. 72 Yamaha, E., Matsuoka, E. and Yamazaki, F. (1988) Nippon Suisan

Gakkaishi 54, 2043. 73 Hallerman, E.M., Faras, AJ., Kapuscinski, A.R. and Guise, K.S. (1990)

Trans. Amer. Fish. Soc. (in press). 74 Inoue, K., Yamashita, S., Hata, J.-I., Kabeno, S., Asada, S., Nagahisa, E.

and Fujita, T. (1990) Cell Differ. Dev. 29,123-128. 75 Maclean, N., Penman, D. and Zhu, Z. (1987) Bioffechnology 5,257-261. 76 Kozlov, A.P., Reshetnikov, V.L., Korzh, V.P. and Neifakh, A.A. (1988)

Molecular BioI. 22, 1286-1293. 77 Inoue, K., Ozato, K., Kondoh, H., Iwamatsu, T., Wakamatsu, Y., Fujita,

T. and Okado, T.S. (1989) Cell Differ. Dev. 27, 57-68. 78 McEvoy, T., Stack, M., Keane, B., Barry, T., Sreenan, J. and Gannon, F.

(1988) Aquaculture 68, 27-37. 79 Rassoulzadegan, M., Leopold, P., Vailly, J. and Cuzin, F. (1986) Ce1146,

513-519. 80 Stinchcomb, D.T., Shaw, J.E., Carr, S.H. and Hirsch, D. (1985) Mol. Cell.

BioI. 5, 3484-3496. 81 Fansler, B. and Loeb, L.A. (1969) Exp. Cell. Res. 57, 305-310. 82 Shioda, M., Nagano, N. and Mano, Y. (1982) Dev. BioI. 91, 111-120. 83 Scott, W.B. and Scott, M.G. (1988) Atlantic Fishes of Canada. University

of Toronto Press. 84 Horvath, L., Tamas, G. and Tolg, I. (1984) Special Methods in Pond Fish

Husbandry (Halver, J.E., ed.) Akaderniai Kiado, Budapest Halver Corporation, Seattle, W A.

85 Vernier, 1.M. (1969) Annales d'Embryologie et de Morphogenese 2,495-520.

86 Wiegand, M.D., Buchanan, L.G., Loewen, 1.M. and Hewitt, C.M. (1988) Aquaculture 71,209-222.

370 G. J. FLETCHER AND P.l. DAVIES

87 Mansueti, AJ. and Hardy, J.D., Jr. (1967) Development of fishes of the Chesapeake Bay region. An atlas of egg, larval and juvenile stages part I (Deubler, E.E., Jr., ed.) Natural Resources Institute, Univ. Maryland, Port City Press, Baltimore, MD.

88 Stickney, R.R. (1986) Culture of non-salmonid freshwater fishes. CRC Press, Inc. Boca Raton, FL.

89 Toner, E.D. (1966) Synopsis of biological data on the pike Esox lucius Linnaeus 1758. F AO Fisheries Synopsis No. 30. 35 pp.

90 Colby, Pol., McNicol. R.E. and Ryder, R.A. (1979) Synopsis of biological data on the Walleye Stizostedion v. vitreum (Mitchill 1818) FAO Fisheries Synopsis, No. 119, 139 pp.

91 Hisaoka, K.K. and Battle, H.1. (1958) J. Morphol. 102, 311-328. 92 Laale, H.W. (1977) J. Fish. BioI. 10, 121-173. 93 Penman, Dol., Beeching, AJ., Lyenger, A. and Maclean, N. (1988) Bull.

Can. Aquaculture Assn. 88-4, 137-139. 94 Tamiya, E., Sugiyama, T., Kazuyoshi, M., Hirose, A., Okoshi, T. and

Karube, I. (1990) Nucl. Acid Res. 18, 1072. 95 Hew, c.L., Wang, N.C., Joshi, S., Fletcher, G.L., Scott, G.K., Hayes, P.H.,

Buettner, B. and Davies, P.L. (1988) J. BioI. Chern. 263, 12049-12055. 96 Scott, G.K., Davies, P.L., Kao, M.H. and Fletcher, G.L. (1988) J. Mol.

Evol. 27, 29-35. 97 Hallerman, E.M., Schneider, J.F., Gross, M., Liu, Z., Yoon, Sol., He, L.,

Hackett, P.B., Faras, AJ., Kapuscinski, A.R and Guise, K.S. (1990) Animal Biotech. 1, 79-93.

98 Ptashne, M. (1988) Nature 335, 683-689. 99 Mitchell, Pol. and Tjian, R. (1989) Science 245,371-378. 100 Huang, R.C.C., Price, J.L. and Gourlie, B. (1990) in Transgenic Models in

Medicine and Agriculture (Church, R.B., ed.) pp. 109-126, Wiley-Liss, Toronto.

101 Friedenreich, H. and Schartl, M. (1990) Nucl. Acids Res. 18,3299-3305. 102 Durnam, D.M. and Palmiter, RD. (1981) J. BioI. Chern. 256, 5712-5716. 103 Byrnes, L. and Gannon, F. (1990) DNA and Cell BioI. 9, 647-655. 104 Scott, G.K., Hew, C.L. and Davies, P.L. (1985) Proc. Nat. Acad. Sci.

U.S.A. 82, 2613-2617. 105 Brinster, R.L., Allen, J.M., Behringer, RR, Gelinas, R.E. and Palmiter,

R.D. (1988) Proc. Nat. Acad. Sci. U.S.A. 85, 836-840. 106 Bonifer, C., Vidal, M., Grosveld, F. and Sippel, A.E. (1990) EMBO J. 9,

2843-2848.

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