Generating biological models through gene transfer to domestic animals

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Generating biological models through gene transfer to

domestic animals

Louis Houdebine

To cite this version:

Review

article

Generating biological

models

_through

_gene

transfer

to

domestic animals

LM

Houdebine

Unité de

_{différenciation}

cellulaire, Inrn, 78352

_{Jouy-en-Josas}

cedex, France

(Received

7

_January

1997;

accepted

23

_January

₁₉₉₇₎

Summary ― Transgenic

domestic animals remain

_infrequently

used as models for biochemical,

biomedical or

_{pharmaceutical}

studies. The

_difficulty

in

_obtaining

these animals and the cost of their maintenance

_explains

this situation. This review

briefly

summarizes the different

_techniques

_{of gene}

transfer,

targeted

or not, and also the

_techniques

for

_constructing

vectors for _transgene

_expression.

A few

_examples

of domestic animal models are also

_reported.

domestic animal /

_transgenic

/ model

Résumé ― L’obtention de modèles

_biologiques

par le transfert de

gène

à des animaux

domes-tiques.

Les animaux

_{domestiques transgéniques}

sont encore _{peu utilisés} comme _{modèles pour des}

études fondamentales ou _pour des

applications

biomédicales et

_{pharmaceutiques.}

La difficulté d’obtention et le coût de ces animaux

_expliquent

en

_{grande partie}

cet état de fait. Cette revue fait le

point

sur les différentes

_techniques

de transfert de

_gène

ciblé ou non _{ainsi que} sur les vecteurs

d’expression

des

_{transgènes. Quelques exemples}

de modèles animaux

_exploités

sont

_également

rap-portés.

animal

_domestique

/

_{transgénique}

/ modèle

INTRODUCTION

About _{15 years ago,}

_pioneering

_experiments

demonstrated the

_possibility

_{of gene}

trans-fer into animals

_resulting

in transmission to

their _progeny as well as

_expression

of the

foreign

DNA

(Gordon

et

al, 1980;

Palmiter

et

al, 1982).

This

_opened

new avenues for

basic studies into _gene function and for

mul-tiple

applications

in medicine and

agricul-ture. New animal models have been created

using

gene transfer and gene inactivation

mainly

with mice

_(Lathe

and

_{Mullins, 1993).}

There are several

practical

reasons for which

mice have been used

_extensively

to

_generate

animal models for the

_study

of diseases.

Gene transfer is

_relatively

efficient in this

species.

Gene inactivation

_{by homologous}

recombination has

_only

been achieved in the mouse, and

finally,

mice can be raised and maintained

_{relatively inexpensively.}

The mouse,

however,

is not

_always

an

appropriate

model. It is too small to be used

easily

for

_{surgical operations.}

It is also too

different from humans in some

_biological

functions. The use of other

_species,

and

par-ticularly

domestic

animals,

is considered

highly

desirable. For technical and finan-cial reasons, this remains limited to a small number of

_situations.

In this

_review,

the dif-ferent

_techniques

of _gene transfer into

ani-mals and a few

_examples

of domestic animal

models are described. Two other reviews

on the same

_subject

have also been

pub-lished

_recently

(Petters, 1994;

Mullins and

Mullins, 1996).

TECHNIQUES

OF GENE

TRANSFER TO ANIMALS Addition of a

_foreign

_gene

The addition of a

_foreign

_gene to a _genome

is

_{theoretically}

the

_simplest

_experimental

modification of the _{genome. To} create lines of

_genetically

modified

animals,

gene

trans-fer must be achieved in the

_{early embryo.}

The transfection

_techniques

used to

trans-fer genes into cultured cells are not

suffi-ciently

efficient to be used for

_embryos.

More

_{sophisticated}

methods must therefore be used in most cases. The direct

microin-jection

of isolated _genes into one of the

pronuclei

of mammalian

_embryos

is the

most

_commonly

used

_{technique. Up}

to five

transgenic

mice can be obtained in this _way

from 100 one-cell

_embryos.

The resultant

yield

is also

_high

in the rabbit but is

_quite

variable in other mammals. The cost of founder

_transgenics

is thus

_high

in

_large

domestic animals

_(Gagne

et

_{a], 1997).}

In

ruminants and

_especially

in cows, one-cell

embryos

can be obtained from

_oocytes

col-lected in the

_{slaughterhouse}

after in vitro maturation and fertilization

_{(Crozet, 1997).}

The cost of

_generating

the

_embryos

is thus

highly

reduced. After _gene

_{microinjection,}

the

_embryo

can

_develop

in vitro until the

blastocyst

stage

when it is transferred into the uterus. This allows the

_spontaneous

elim-ination of

_embryos

which do not survive after

_{microinjection}

and

_possibly

the selec-tion of those which harbour the

_foreign

_gene

(Thompson

et

al, 1995).

This

_approach

has the

_advantage

of

_{considerably reducing}

the number of

_recipient

females and therefore the cost of the

_transgenic

founders.

In lower vertebrates and invertebrates

injection

into the

_cytoplasm

of

_embryos

_may

generate

transgenic

animals

_(Devlin,

1997).

Use of viral vectors

Defective viral _genomes can transfer

for-eign

genes into

embryos.

This is

particu-larly

the case for retroviral vectors

_(Ronfort

et

al, I 997).

These vectors have a

_specific

or

amphotropic envelope

and show limited

efficiency. They

are

_only

used in birds and

invertebrates.

Somewhat

_{unexpectedly,}

recent

experi-ments have shown that

_{non-replicative}

ade-noviral vectors

_integrate

into the mouse

embryo

genome with considerable

effi-ciency (Tsukui et

al,

1996).

Use of

_embryonic

cells

Totipotent embryonic

stem cells

_{(ES cells)}

can be cultured for

_{long periods}

of time and

participate

in the

_development

of chimaeric

embryos

after

_having

been

_injected

into the

embryos

at the morula or the

_blastocyst

stage.

Gene transfer can be carried out in these

trans-fection methods.

_{Multiple sophisticated}

vec-tors make it

_possible

to select the

_totipotent

cells which have

_integrated

the _gene

_through

an

_homologous

_{recombination process. This}

allows

_specific

_{gene knock-out} or

replace-ment (Viville, 1997).

In

_{practice, totipotent}

cells can

_only

be

used in the mouse. Recent

_experiments

indi-cate

_{that totipotent}

cells have been obtained

in the

_pig

_{(Anderson, 1996)}

and chicken

(Pain

et

_{al, 1996).}

Interestingly,

sheep multipotent

cells lines

incapable

of

_{participating}

in the

_generation

of chimaeric

_embryos

can result in the birth of normal lambs after

_being

introduced into

enucleated

_oocytes

_(Campbell

et

_{a], 1996).}

This

_experiment

offers

_{quite interesting}

pos-sibilities if the

_techniques

can be

_improved

and extended to other

_species

and if

_foreign

genes can be introduced into these cells for gene

targeting

or for

_{simple transgenesis.}

Spermatozoa

precursors are also

_potential

vectors for

_foreign

_{gene transfer. In vitro} fertilization can be achieved with immature

spermatozoa

using

microinjection

into

oocytes.

Mice testes can also be colonized

with immature

_spermatozoa

from other

mice,

rats and

_potentially

other

_species.

These

_spermatozoa

then become

_fully

func-tional

_{(Clouthier et al, 1996).}

Conventional

or

_targeted

_gene transfer could be carried

out

_during

the culture of the

_spermatozoa

precursor.

Although promising,

these

tech-niques

cannot be used at

_present

_{for gene}

transfer.

_Improvement

of cell culture

tech-niques

is

_required

before it becomes

possi-ble.

VECTORS FOR TRANSGENE EXPRESSION

Gene transfer

_by

transfection or

microin-jection

leads to uncontrolled

_integration

and

to an

_{unpredictable}

level of

_transgene

expression.

Gene insulators that allow a

_high

and copy number

dependent expression

of

transgenes

have been identified

_(Sippel

et

at,

1997).

Full characterization of these insu-lators is still

_required

before their use can

be

_generalized.

In some _cases,

_{large genomic}

DNA

_fragments

contain insulators

_(Umland

et al, I 997).

Gene knock-out is not the

_only

_way to

specifically

inactivate _gene

_expression.

Anti-sense RNA and

_ribozymes

can reduce or

inhibit the

_synthesis

of a

_{protein by}

inacti-vating

its mRNA

_{(Carmichael, 1997;}

Han

and

_Wagner,

_1997).

_{Overexpression}

of

mutated genes

coding

for

_{proteins having}

dominant

_negative

effects can also be used

successfully

in some cases

_(Chang

et

ai,

1994).

Non-secreted antibodies

(intrabod-ies)

may also act

_efficiently

to inactivate cellular

_proteins

_(Jones

and

_{Marasco, 1997).}

Promoters reconstituted

_by

_genetic

engi-neering

can be sensitive to inducers

_having

no effect on _{host genes. These} inducers can

switch

_transgenes

on and off in a

_specific

and

_potent

manner.

_{Tetracycline-sensitive}

promoters

are _among those which have met

with

_impressive

success

_(Kistner

et

_at,

1996).

Quite

specific

and efficient recombina-tions can be induced at

_specific

DNA sites

by foreign

recombinases. The most

fre-quently

used

_system

is based on the

utiliza-tion of a lox DNA _{sequence and} on the Cre recombinase from the E coli

_{P !}

_phage.

The

Cre recombinase

_triggers

a recombination

of the lox sequences which may be intro-duced into known sites on _gene constructs or on _{genomes. This recombination may lead} to

the

_specific

inactivation of a _{gene if} it

gen-erates a _{deletion within the gene.} It _may activate a

_promoter

as well

_{by removing}

an

inhibitory

DNA

_fragment.

_{It may also lead}

to

_homologous

recombination and the

tar-geted

knock-out of a

_given

cell

_type

at a

given

stage

in its differentiation. This

promoter

specifically

active in this cell

(Kuhn et al, 1995).

There are _{many other}

_possible

combina-tions of these tools which result in a

rea-sonably satisfactory

control of

_transgene

expression.

TRANSGENIC DOMESTIC ANIMAL MODELS

Apart

from the mouse, the rabbit is

probably

y the most

_frequently

used

_species

as an

ani-mal model. Rabbits are as sensitive to

atherosclerosis as humans. Human

apolipoprotein

genes have been transferred

to rabbits

_(Duverger

et

al,

1996;

Mc

Cormick et

_{al, 1997).}

These animals arc more resistant or sensitive to

cholesterol-rich diets than control rabbits

_depending

on

the

_particular

_{genes transferred.}

_They

are

good

models for

_studying

_{the process of}

atherosclerosis,

for

_evaluating

the

_efficiency

of the

_drugs

that stimulate the

_expression

of _{the genes}

_coding

for

_{high density}

lipopro-teins and for

_testing

_gene constructs that may be used in gene

therapy.

Moreover,

these

_transgenic

_{rabbits may be crossed with} the Watanabe line which is devoid of low

density lipoprotein

(LDL)

receptors

and so

may

provide interesting

information

con-cerning

the effect of

_{apolipoproteins}

on

atherosclerosis.

Rabbit cells can

_generate

Human

Immunodeficiency

Virus

_(HIV)

_particles

when transfected with the viral _genome.

Transgenic

rabbits

_expressing

the human CD4 gene can be infected with HIV. These

animals did not _go on to

_develop

AIDS but

they

may be

good

models to evaluate the

efficiency

of a vaccine

_(Dunn

et

_{al, 1995).}

The use of

_pig

_{organs for}

_{transplantation}

to humans is

_{becoming increasingly}

neces-sary. The

study

of the

hyperacute

rejection

of pig

organs can be carried out with

trans-genic pigs expressing

various

_{foreign genes.}

The _organs from

_pigs

_expressing

human

DAF or CD59 genes are

_rejected

much more

slowly

than those from control animals

(Cozzi

and

White,

1995).

The number

_{of transgenic}

domestic

ani-mals raised and used as models for

biolog-ical,

biomedical or

_{pharmaceutical}

studies is

still very low

although

their use is

_easily

justified.

This is

_largely

due to the technical difficulties involved and the cost of

obtain-ing

these animals. Substantial _{progress is}

being

made on the

_manipulation

of

_embryos

and

_embryonic

stem

_cells,

and ttlso on the

construction of efficient vectors for

trans-gene

expression.

The

_generation

of domes-tic

_transgenic

animals should therefore

become more feasible in the

_coming

_years.

It should be

_kept

in

mind, however,

that

generation

and

_breeding

of

_{large transgenic}

animals will remain a

_{relatively demanding}

task. This

_implies

that the animals lines used for

_transgenesis

be chosen

_carefully.

Indeed,

the

_{genetic background}

_{of the animals may}

have

_quite

a

_significant

_impact

on the effects

of the

_transgene

and

_impair

the

_validity

of

the models

(Carvallo

et

at, 1997).

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