SUISANZOSHOKU

49(2), 137-142(2001)

第4回

日韓 ・韓 日水産増養殖 シンポジウム基調講演

Review

Gene

Transfer

in Salmonidae:

Applications

to Aquaculture

Goro YOSHIZAKI*1 (Accepted May 17, 2001)

Abstract: Transgenic technique has been applied to commercially valuable species in recent years and is expected to be a powerful approach for fish breeding. This brief review describes the status quo and discusses the future prospects of transgenic research in salmonidae. The mainstay of this research has been the successful microtechnique whereby a foreign gene is transferred into the blastodisc of a fertilized egg, with the aid of a micropipette. A portion of the microinjected genes is retained in the host cell, integrated into the host chromosome, and expressed before being inherit -ed by subsequent generations. Based on these principles, recently, a Canadian group produced transgenic salmon showing rapid growth by introducing foreign growth hormone gene. In other studies the introduction of freezint resistance and low dissolved oxven tolerance have been attempted. -five

way

are

also

Besides of producing

this,“cell-mediated transgenic

gene fish.

The

transfer”has benefits

evolving

applying

as

transgenic

a convenient research

and

effec

in aquaculture

highlighted.

Key words:

Transgenic

fish; Salmonidae;

Gene;

Transgenic fish research has been booming, thanks to zebrafish and medaka, which serve as model specimens in basic biology studies. This technique has also been applied to commercial -ly valuable species in recent years and is expect -ed to be a powerful approach for fish breeding. In the field of aquaculture, domesticated strains that have desirable genetic traits (such as those possessed by other terrestrial animals, e. g., Holstein cow, Yorkshire pig, white Leghorn chicken) are very few because the history of rearing aquatic species on scientific principles is not as long as that of animal husbandry, and conventional selective breeding techniques take a long time to obtain such strains. On the other hand, molecular and cellular biological tech - niques may be rapid and effective in improving genetic characters of cultured fish. In this review, I introduce the recent progress of transgenic techniques in salmonidae including a new gene *1 Laboratory

of

been

of Aquaculture

Tokyo 108-8477, Japan.

, Department

Biotechnology

transfer

method

- fer”and

their

called“cell-mediated future

aquacultural

gene

trans

applications.

1) The technique Foreign genes can be introduced into salmonid eggs by microinjection, a technique that allows the introduction of a small volume of foreign gene solution into the blastodisc of a fertilized egg by a very thin glass pipette (micropipette). However, a problem had to be circumvented when applying this technique to salmonid eggs. Although the chorion of an unfertilized egg is very soft and easily penetrated by glass micropipette, that of a fertilized egg becomes very hard after water activation (Fig. 1) and is a big barrier for microinjection. It is no joke that one fertilized egg can hold up to 1 kg weight on it at 16 hrs after water activation, let alone attempting microinjection. To overcome this dif - ficulty, several methods for microinjection have

of Aquatic Biosciences,

Tokyo University

of Fisheries,

4-5-7 Konan, Minato-ku,

G. Yoshizaki

138

been developed. Chourrout et al.1) pierced the chorion with a metal needle and subsequently introduced the foreign gene into the egg through the hole using a glass micropipette. Although the time span for the operation is lim -ited, microinjection can be done just after water activation and prior to hardening of the chori - on2). Our approach to the problem was some - what different and we found that the hardening of rainbow trout eggs can be postponed effec -tively by treating with reduced glutathione solution (Fig. 1, broken line), facilitating easier microinjection3,4). After this treatment, approxi - mately 107 copies of foreign genes suspended in 2 nl Tris-EDTA buffer are microinjected into the cytoplasm of 1-cell stage eggs (Fig. 2). By this method, most of the microinjected eggs develop normally and circa 50%of the embryos stably carry the foreign gene in their chromo -somes5). For effective application in aquacul -ture, broodstock fish carrying the foreign gene homozygously that can be produced at F2 gen - eration should aid the mass production of trans - genic fish through simple artificial fertilization procedures. These foreign genes integrated in the host chromosomes are expressed under the control of regulatory elements, such as the pro - moter and enhancer. To achieve the tissue-or stage-specific expression of foreign genes, those elements derived from fish, especially from close-

Fig. 1.

Effect trout

of

glutathione

chorion

after

insemination,

eggs

glutathione spring

solution

was

weights

- ed

on

on the

(pH

the

an

egg

8.0)

Y-axis.

The

in 1 (broken

line)at by

at that

the

10℃. applying

various

time

points

could

break

the

values

hardening

Following

activated

measured

weight

on

fertilization. were

water(control;solid

chorion

- mining

treatment

were

averages

ly related species are preferable 2) Its' aquacultural

(see below).

applications

In 1986, the production of transgenic trout carrying foreign growth hormone gene was first reported by Chourrout et al.1). Since then, various researchers have reported their trials using rainbow trout, Atlantic salmon, Chinook salmon and coho salmon6). Most of the studies in salmonidae attempted to produce transgenics carrying foreign GH genes that enhance their growth rate. The early results were not encour - aging, suggesting that the use of promoter and growth hormone genes isolated from higher vertebrates and their heterologous sequences were not fully functional in salmonid species. However, Devlin's group reported that all-fish construct containing salmon growth hormone gene driven by salmon metallothionein promot - er or ocean pout anti-freeze protein promoter stimulated growth of various salmonid species very effectively7). The most rapid growing salmon grew more than 10 times faster com - pared to non-transgenic siblings8). Recently, a private Canadian company, named AquaBounty Farms has been working on these transgenic fish to collect various physiological data to veri -fy their suitability for human consumption.9,10,11) Another interesting trial has been the introduc -tion of cold tolerance into salmon12). On the Atlantic coast of Canada, sea-cage culture of salmon is restricted to relatively small areas of the Southern coast during the winter season, because the other areas have sub-zero water temperatures. However, several species living in

of

artificial mM

reduced line)or

in

Hardness

of

increasing and egg, of

deter indicat 10

eggs.

Fig. 2. Foreign gene microinjection into a blastodisc of a fertilized egg.

Transgenic

such icy seawater in high latitudes avoid freez -ing their blood and survive even at sub-zero temperatures, probably by producing anti-freeze proteins (AFP). Fletcher and Hew's group has been working on the creation of transgenic, freeze-resistant salmon using the AFP gene. AFP genes were isolated from winter flounder and were microinjected into the fertilized eggs of Atlantic salmon. It was successfully integrat - ed into the host genome, transmitted to off - spring and expressed. However, the expression level of AFP was not high enough to increase the freezing resistance of the salmon13). We have been studying a transgenic trout that car -ries the carp globin gene4,14).Carp hemoglobin can supply oxygen to tissues under low oxygen conditions, while trout hemoglobin can do so only under oxygen-rich conditions. The aim is to develop a transgenic trout that will be able to tolerate low dissolved oxygen environments. Further research will be needed to confirm the phenotype of these transgenic trout.

Salmon

139

1) Establishment of stem cell line

2) Gene transfer and cell selection

3) Germ-line chime production Fig.3.

Schematic - duce

-jection; however, the establishment of a stable transgenic line, especially, the production of homozygous transgenic trout with respect to foreign gene requires a lot of labor, space, time and cost. This calls for the need of establishing a new technique to produce transgenic fish line for farmed fish species that requires a lot of space to raise and long time to mature. With this objective, we have been recently trying to develop a stem cell-mediated gene transfer method in rainbow trout. This approach follows 3 major steps (Fig. 3) : I) the establishment of stem cell lines which have the ability to differ -entiate to germ cells; II) the introduction of for -eign gene into the cultured stem cell and selec -tion of transgenic cells using selectable markers such as a drug resistance gene; and III) the con - version of transgenic stem cell to individual fish via germ-line chimera fish production. The germ-line chimera is produced by transplanta -tion of the transgenic stem cells into developing embryos. The fish hatched out from these eggs

fish

-fer”. Foreign cells

that

These fish

individuals

and

gray

are

an

via

cells,

-ing

shows

consists the

egg

cultured - eage,

line

cultured

germ

cells.

converted

chimera.

into

White and

Black

and

transplanted

cultured

cells

transgenic

gray

gamete)derived

of cells-those

and

those

from

fishes(or from cells

carry

gonad

derived the

cells have

into the germ

of chimera

from

transplanted

if the cultured

to differentiate

the

are

fishes(or

cells. Further,

an ability

the to

pro

trans

respectively.

of 2 types

host

into

cells

to

gene

non-transgenic

and

genes,

strategy

differentiate

respectively.

eggs

foreign

to

germ

gamete)represents host

the

transferred

ability cultured

cells

cultured

of

using“cell-mediated

genes have

transgenic

the

3) A novel approach (cell-mediated gene transfer) As described above, we have developed a method to produce transgenic trout by microin

representation

transgenic

fish

cell lin

can

contain

gametes derived from the transplanted cultured cells. This means that if the eggs and sperms that are derived

from the cultured

to fertilize

each

completely

derived

cells. this

One

of the

method

- duced

into

carrying easily

other, most

is that stem

foreign

genes

in a small

resulting

the

in

vitro

petridish.

are stem

advantage

genes

can

fish

transgenic

important

cells

foreign

the

from

cells are used

are

and

be By

the

selected the

of

intro cells very

standard

gene-transfer method, we have to rear a lot of fish and analyze a huge number of DNA for transgenic

candidate

screening

seems

technique - duction

to be a short

is a powerful of foreign

sequence The

fish, whereas,

recent

genes

modification progress

this in vitro

cut. Further,

this

tool not only for intro but also for nucleotide of endogenous

in protein

engineering

genes. has

140

G. Yoshizaki

Fig. 4. Trunk region of a transgenic trout embryo carrying green fluorescent protein (GFP) gene driven by vasa gene promoter. PGCs indicates primordial germ cells showing specific green fluorescence. I; intestine showing autofluorescence.

revealed that modification of a few amino acid sequences often up-regulate the activity of pro -teins. Therefore, the modification of endoge - nous gene would change the phenotype of fish individual and could be used as a new method for fish breeding. As a stem cell used for this method, we have chosen primordial germ cell (PGC) that is a precursor cell of germ cell lineage. In fish, a technique to identify and isolate live PGCs was lacking. In rainbow trout, we proved that the distribution of vasa gene transcripts is restricted to the germ cell lineage, making this transcript a useful indicator of PGCs15). This means that the regulatory regions of rainbow trout vasa-like gene (RtVLG) are activated only in PGCs. Therefore, we cloned and character -ized the rainbow trout vasa-like gene (RtVLG) regulatory regions and produced transgenic trout carrying the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (pvasa-GFP)16). GFP is a protein isolat -ed from jellyfish and shows strong green fluo -rescence without any co-factor. Therefore, it has been widely used as a cell labeling tool and a marker for foreign gene expression17). This transgenic trout carrying pvasa-GFP expressed GFP gene specifically in PGCs. Consequently, these trout showed specific green fluorescence in PGCs (Fig. 4). These trout embryos having

Fig.

5.

Primordial

trout

having

cytometer. -rescence

germ

cells

GFP-labeled

Left and right field picture,

isolated germ panel

show

from cells

transgenic using

bright

flow

and fluo

respectively.

the GFP-labeled PGCs were dispersed using trypsin and GFP-positive germ cells could be isolated using their fluorescence as an indicator with a flow-cytometer (Fig. 5). The in vitro culture of these PGCs and gene transfer into the PGCs are now under progress. We are cur -rently trying to develop a modus operandi to transplant isolated PGCs (and in vitro-cultured PGCs) into early embryos to produce germ-line chimeric trout to obtain fish derived from cul -tured PGCs using a cell microinjection method developed by our laboratory18). Thus, visualiza -tion and isolation of germ line stem cell has been achieved and the cell-mediated gene trans -fer method would become available in the near

Transgenic

future

using

these

techniques.

Salmon

141

suggested

that it is possible

amount 4)Future

aspects

In

this

review,

transgenic

only

salmonid the

this

technique

to

For

instance,

disease

and

be

other

important

also

functions, - high - duction

possible

such

as

suppress

genes

cancer

and

proposed

as method

and

potentially

words,

easier fish

pharmaceuticals”.

with

without

is

that

In

fact,

large

amount

Fig.6

shows

foreign

driven

by

Fig.7is

a

strong picture

of

muscle

- orescence

a

in

piece

of

cells)showing

caused

by

no-less

important

of the

and

Transgenic gene in

driven the

- transgenic

center

rainbow by is

Medaka transgenic

siblings.

trout

β-actin and

of these

to accidentally

To prevent

wild fish

re-modeled

sum

advan

potential

between

risk

escaped

population,

was made

flesh

- evant

inac

the

a

advances

possible

these

techniques

- al importance

popu

transgenic

usage

of sterile

in closed

in salmonid

The

in other would

re

considered.

physiological

of this family.

if

of repro

To

research

due to the abundance

background

species

foreign

flesh.

up, these

fish

mix with natural

the

interaction

eco

in rel

information

on

cross-application species

of

of aquacultur

not be a surprise.

carrying

Acknowledgements

promoter

trout

muscle. trout

flesh

green

flu

I gratefully the

gift

of

others

acknowledge the

Dr.

Medaka

M.

Kinoshita

β-actin/GFP

for

construct.

results

carrying

promoter. the

impact

on the

potential

on-going

These

embryo

aspect

of

in science

with research

Fig.7.

Fig.6.

advances

be complemented

them

fish

the

gene.

These

production

be

strong

GFP

fish.

expression

should

- ductive

produced

β-actin

activity

quality

for effective

rearing

embryos

Medaka

desirable

the to be

further knowledge has controlling phenotypic

should

raw

in

precisely. Besides this, to be gained on genes

gene

system

the

protein trout

foreign

need

fish and

otherwise

successfully

the

the mole

of fish. Especially,

expression

transgenic

the

from

biology

for

to aquacul

about

- circulation

one

eat

information

of gene

they were

other

in edible

techniques

as“edible are

would

have

of

shows

(mostly

can

transgenic

gene

which

we

produced

we

used

more

to control

-lations.

use.

In

however,

it, which

protein

gene.

GFP 20)

fish

the

be

be

cheaper

administer.

and cellular

- biological

can

human

trials

crops19);

heating

- ovate

for

can

that

pressure,

vaccination

Similar

terrestrial

-tage

to

intro

fish

- cular

high

anti

by

we need

characters

proteins

vaccines oral

it

and

fish

high-blood

make

transgenic

using

activity into

transgenic

edible will

studied.

non-piscian

encoding

Further,

This

introduce

- ture,

studied

research,

proteins

to apply these

mechanisms

resis would

be of

activity,

foreign

respectively.

enormous.

to

anti-cancer

In order

applying

quality

realms to

pressure

of

flesh

of pharmaceutical

a large

part of fish by introduction of appropriate - eign genes instead of the GFP gene.

intro

stress

issues the

be

blood

are

resistance, of

of

been of

aquaculture

extending

would

have

possibilities

improvement

Further,

examples

research

- duced;however,

-tance,

selected

to produce

GFP The are

one non

Cross

section

and

transgenic

gene

driven

panel

is a bright

- orescence strong

by

picture. green

of

1-year

rainbow Medaka field

old

non-transgenic

(left)

trout(right)carrying β-actin picture

Flesh

fluorescence.

of

and

GFP

promoter. the

transgenic

right trout

The one

left is flu

showed

G. Yoshizaki

142

genic Atlantic salmon (Salmo salar). Aquaculture,188, 47-63. 12) Fletcher, G. L., M. A. Shears, M. J. King, P. L. Davies, and C. L. Hew (1988): Evidence for antifreeze protein gene transfer in Atlantic salmon. Can. J. Fish. Aquat. Sci., 45, 352-357. 13) Shears, M. A., G. L. Fletcher, C. L. Hew, S. Gauthier, and P. L. Davies (1991) : Transfer, expression and sta - ble inheritance of antifreeze protein genes in Atlantic salmon (Salmo salary. Mol. May. Biol. Biotechnol., 1, 58-63.

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Yoshizaki,

G.,

Introduction Nippon

of Suisan

T.

O shiro,

carp

and

α-globin

F.

14)Yoshizaki,

in

rainbow

globin

Devlin,

R.

H.(1997):Transgenic

“Transgenic

animals,

M.Houdebine),

generation Harwood

Amsterdam,

salmonids, and Academic

use”(ed.

F.

gene

line introduced

Takashima,

I. Hirono,

transmission

in rainbow

of

trout.

L.

Publishers,

pp.105-122.

7) Devlin, R. H., T. Y. Yesaki, E. M. Donaldoson, S. J. Du, and C. L. Hew (1995): Production of germline trans - genic Pacific salmonids with dramatically increased growth performance. Can. J. Fish. Aquat. Sci., 52, 1376-1384. 8) Devlin, R. H., T. Y. Yesaki, C. A. Biagi, E. M. Donaldson, P. Swanson, and W. K. Chan (1994) : Extraordinary salmon growth. Nature, 371, 209-210. 9) Cook, J. T., M. A. McNiven, G. F. Richardson, and A. M. Sutterlin (2000): Growth rate, body composition and feed digestibility/conversion of growth-enhanced transgenic Atlantic salmon (Salmo salar). Aquaculture, 188,15-32. 10) Cook, J. T., M. A. McNiven, and A. M. Sutterlin (2000): Metabolic rate of pre-smolt growth-enhanced transgenic Atlantic salmon (Salmo salar). Aquaculture, 188, 33-45. 11) Cook, J. T., A. M. Sutterlin, and M. A. McNiven (2000): Effect of food deprivation on oxygen consump -tion and body composition of growth-enhanced trans-

carp

Nippon

20)

Hamada, Y.

K.,

K.

Wakamatsu,

medaka GFP latipes).

Tamaki, and

β-actin reporter Mol.

T.

K.

promoter gene

Mar.

Sasado,

Ozato(1999):

Y.

investigated in

Biol.

transgenic Biotechnol.,

Watai,

S.

Usefulness using

され て い る 。 本 総 説 で は,サ ケ 科 魚 類 にお け る遺 伝 子 導 入 技 法 の 最 近 の 進 展,お

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下 の 海 で も養 殖 可 能 な サ ケや,低

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the

a mutant

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Suisan

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込 まれ,発

and

15) Yoshizaki, G., S. Sakatani, H. Tominaga, and T. Takeuchi (2000) : Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage. Mol. Reprod. Develop. 55, 364-371. 16) Yoshizaki, G., Y. Takeuchi, S. Sakatani, and T. Takeuchi (2000): Germ cell-specific expression of green fluorescent protein in transgenic rainbow trout under control of the rainbow trout vasa-like gene pro - moter. Int. f. Dev. Biol. 44, 323-326. 17) Takeuchi, Y., G. Yoshizaki, and T. Takeuchi (1999): Green fluorescent protein as a cell labeling tool and a reporter of gene expression in transgenic rainbow trout. Marine Biotechnology, 1, 448-457. 18) Takeuchi, Y., G. Yoshizaki, and T. Takeuchi (2001): Production of germ-line chimeras in rainbow trout by blastomere transplantation. Mol. Reprod. Develop. (in press). 19) Fischer, R., J. Drossard, U. Commandeur, S. Schillberg, and N. Emans (1999): Towards molecular farming in the future: moving from diagnostic protein and anti - body production in microbes to plants. Biotechnol. Appl. Biochem., 30,101-108.

in By

Oshiro,

Gakkaishi,57,2203-2209.

trout.

Gakkaishi,57,819-824.

5) Yoshizaki, G., S. Kobayashi, T. Ochiro, and F. Takashima (1992): Introduction and expression of CAT gene in rainbow trout. Nippon Suisan Gakkaishi, 58,1659-1665. 6)

T.

T.Aoki(1991):Germ

Takashima(1991):

gene

G.,