D9205.PDF

Rev. sci. tech. Off. int. Epiz., 1 9 9 8 , 1 7 (1), 365-378
Transgenic approaches to the increase of
disease resistance in farm animals
M . Müller & G. Brem
Institute of Animal Breeding and Genetics, Veterinary University of Vienna, Veterinärplatz 1, A-1210 Vienna,
Austria; Department of Biotechnology in Animal Production, Institute for Agrobiotechnology Tulln,
Konrad-Lorenzstrasse 20, A-3430 Tulln, Austria
Summary
Molecular genetics and reproductive biology techniques enable the transfer of
foreign D N A into m a m m a l s . Novel a p p r o a c h e s t o modify disease resistance or
s u s c e p t i b i l i t y in l i v e s t o c k a r e j u s t i f i e d b y e c o n o m i c a n d a n i m a l w e l f a r e c o n c e r n s .
Current research on t h e improvement of disease resistance by gene transfer
f o c u s e s on t h r e e m a i n s t r a t e g i e s , as f o l l o w s :
a) s o m a t i c g e n e t r a n s f e r , i.e., n u c l e i c a c i d v a c c i n e s
b) d e l e t i v e g e r m - l i n e g e n e t r a n s f e r , i.e., g e n e k n o c k o u t
cl a d d i t i v e g e r m - l i n e g e n e t r a n s f e r .
These strategies aim at either the transient or stable expression of c o m p o n e n t s
known t o influence non-specific or specific host defence mechanisms, or the
disruption of genes k n o w n t o cause susceptibility t o disease. Referring t o t h e
s o u r c e of t h e effective a g e n t a n d t h e site a n d m o d e of a c t i o n , t h e s t r a t e g i e s are
termed 'genetic', 'congenital', 'intracellular' a n d 'extracellular' immunisation.
Each gene transfer experiment has t o be evaluated carefully w i t h r e s p e c t to the
potential to create novel cases of p a t h o g e n resistance o r t o lose species barriers
or cell-type restrictions.
Keywords
Disease resistance - D N A vaccine - Gain of f u n c t i o n - Gene constructs - I m m u n i s a t i o n Loss of f u n c t i o n - Transgene.
Introduction
research. Relevant biological homologues of human diseases
The pioneer experiments which demonstrated that stable
gene transfers into mice, gene expression and a phenotypic
effect of the transgenes were possible date back more than
fifteen years ( 3 6 , 3 7 , 7 0 ) . The production of transgenic farm
animals - rabbits, pigs and sheep - was reponed in 1 9 8 5 ( 1 4 ,
41). In the following years, this novel technique was also
successfully applied to goats and cattle (16).
plethora of models generated by gene transfer experiments
and therapeutics are also created by gene transfer. In mice, a
resulting in either gain or loss of function are available (7, 8,
69). Transgenic farm animals are ready to be used as the
source
of
recombinant
pharmaceutical
of
high
purposes
value
('gene
for
fanning',
'nutraceuticals') ( 1 5 , 2 4 , 3 0 ) . Recently, very encouraging
experiments
demonstrated
xenotransplantation.
Gene transfer, offers the prospect of developing completely
new breeding strategies and novel applications. The use of
transgene technology in agriculture for animal production
remains very limited compared to the various transgene
approaches used in plant production. This is due to the
technical problems which persist in the generation of
transgenic farm animals at reasonable costs. One general
observation is that the 'bigger' the mammal, the 'harder' it is to
successfully transfer gene constructs. The real success of
transgenic mammals is undoubtedly in the field of basic
proteins
or nutritional
The
the
hyperacute
feasibility
rejection
of
reaction
caused by the primate complement was greatly delayed by
expressing complement inhibitors in grafted transgenic pig
hearts. Therefore, transgenic pigs expressing the human genes
downregulating the complement systems and other genes
counteracting the graft rejection reaction might serve as organ
donors for human recipients (27, 7 2 ) . The induction of new
genetic traits in domestic animals by transgenesis, however,
still awaits success. This is partly due to the technical
difficulties but is mainly caused by the lack of valid identified
genes. In this paper, the authors focus on the methods of
366
Rev. sci. tech. Off. int. Epiz., 17 (1)
transferring genes into farm animals and the possibility of
introducing disease resistance traits by this technique.
Gene transfer methods
Gene transfer performed in early embryonic stages aims at the
integration of the gene construct into all tissues including the
germ line, and is therefore termed 'germ-line gene transfer'.
The technique requires profound skills in reproductive
technologies, i.e., embryo collection or production, embryo in
vitro culture, embryo manipulation and embryo transfer. The
reproductive techniques are reviewed extensively elsewhere
(9, 16). Somatic gene transfer is applied to neonates at the
earliest and results in transient gene expression which lasts
from a few days to the entire lifespan of the recipient.
Germ-line gene transfer
Three main routes to germ-line transgenesis in mammals have
been described and used routinely, as follows:
a) integration of (retro)viral vectors into an early embryo
b) microinjection of DNA into the pronucleus of a fertilised
oocyte
c) incorporation of genetically manipulated embryonic stem
(ES) cells into an early embryo or the use of transgenic
embryo-derived cells in cloning by nucleus transfer.
Gene transfer by microinjection of DNA constructs into early
stage embryos still remains the preferred method for
generating transgenic large animals ( 1 6 ) . The DNA is
microinjected in buffered solution direcdy into the
pronucleus of a fertilised oocyte. The pronuclei of zygotes of
some species, e.g., pigs, have to be visualised by centrifugation
since the cytoplasm is Riled with granula. The equipment
required for routine gene transfer programmes by
microinjection is listed elsewhere ( 1 3 , 16). Depending on the
species used for gene transfer into farm animals, the efficiency
(transgenic neonates/microinjected zygotes) of the technique
routinely reaches 0 . 5 % - 3 % . Data collected during a gene
transfer programme indicating the success rates in the
differing species are given in Table I.
Due to safety considerations and the lack of gross advantages
in gene transfer efficiency, retroviral vectors are not
commonly used. Nevertheless, efforts are being undertaken to
develop vector and delivery systems which fulfil the safety and
efficiency criteria ( 7 7 ) . Other methods, such as using
liposomes as a DNA vehicle or techniques originally
developed for the transfection of eukaryotic cells, have not yet
been established for practical use (13). In 1 9 8 9 , Lavitrano et
al. attracted widespread attention by reporting the production
of transgenic mice by mixing DNA with spermatozoa prior to
in vitro fertilisation ( 5 2 ) . Attempts to establish spermmediated gene transfer as a routine method in other
laboratories have failed to date (80), despite the existence of
other reports in addition to the original publication. The
reason for this remains unclear. The type and secondary
structure of the DNA, the incubation buffer and intermediate
factors (embryonic retroposon activity) are discussed as
crucial factors. However, the fact that sperm cells are able to
take up exogenous DNA spontaneously and transfer it to the
oocytes during the process of fertilisation, thereby causing
transgenesis in a proportion of the resulting embryos, is well
established. This technique was applied to ejaculated semen
and resulted in transgenic adult pigs and transformed bovine
blastocysts ( 8 4 ) . The underlying processes at the molecular
level are currentiy under investigation ( 5 9 , 1 0 7 ) .
A variety of ES cell-like cell lines have been reported in species
other
than mouse.
However,
successful
germ-line
transmission of these cells has not been reported for any of
these species (47, 69). The recent progress in cloning animals
by nuclear transfer might make the establishment of ES cells
in farm animals unnecessary ( 2 0 , 1 0 2 ) . The generation of
viable lambs was achieved from the transfer nuclei of
embryo-derived epithelial cell lines to an enucleated
metaphase-II-arrested
oocyte.
Further
experiments
demonstrated the feasibility of genetically modifying the
embryo-derived cell lines and the subsequent generation of a
transgenic lamb by nucleus transfer cloning (81). Recently a
group of scientists in the United States of America (USA)
reported the use of this technique in cattle (75).
Table I
Generation of transgenic mammals: a comparison of species
Gene transfer statistics
Mouse
Rabbit
Swine
Sheep/goat
Cattle
Number of zygotes per donor
15
70%
20
60%
15
4
5
40-60%
50-80%
10-20%
15%
10%
5%
40-60%
15%
10%
2-8%
1-5%
10-15%
0.5-1%
Pregnancy rate after embryo transfer
Neonates/embryos transferred
Transgene integration frequency
Gene transfer efficiency
la|
,b|
a) Transgenic animals/neonates
b| Transgenic animals/injected embryos transferred
10%
5-10%
5-10%
1-2%
0.5%
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Rev. sci. tech. Off. int. Epiz., 17 (1 )
Somatic gene transfer
Somatic gene transfer does not aim at the germ-line
integration of the transgenes. Its main application is the field
of gene therapy in humans. The methods available for
delivering DNA into differentiated mammalian cells can be
divided into viral and non-viral methods ( 6 2 ) . Viral vector
systems have the advantage of being able to enter cells
actively: however, they face problems concerning safety and
efficiency. The latter problem is mainly due to transcriptional
shutdown and low-infectivity of quiescent cells ( 9 5 ) . The
non-viral methods use direct injection of purified ('naked') or
bound DNA into the blood stream, muscles or other organs
and tissues. Particle bombardment with DNA bound to
microparticles (commonly gold or wolfram, referred to as the
'gene gun') represents an analogous method for injecting
genes into the nucleus of target cells. Jet injection refers to the
acceleration of DNA solutions for the delivery to the
epidermis ( 3 4 ) . Certain methods for controlled delivery of
small-molecule drugs or proteins may be useful for delivering
genes to cells: these include the binding of DNA to cationic
lipids, liposomes, endosomal release peptides, targeting
ligands and polymeric carriers (20).
Enhancement of disease
resistance by transgenic means
Increased disease resistance can be achieved by introducing
resistance-conferring gene constructs into animals or by
depleting a susceptibility gene or locus from the animal.
Hence gain of function (additive) as well as loss of function
(deletive, knockout) gene transfer experiments can be used.
The mechanisms of disease resistance or susceptibility are
frequently controlled by complex genetics and are strongly
influenced by environmental
effects.
Gene
transfer
experiments are often hampered by the lack of identified
major genes or loci responsible for resistance traits. Resistance
or susceptibility mechanisms may occur at all levels of the
pathogen-host interaction, i.e., pathogen entry of the host,
distribution in the host, attachment to host organs, tissues and
cells, possible penetration into cells, multiplication of the
pathogen, maintenance of the pathogen in the host organism
and release of the progeny of infectious agents. Candidates for
gene transfer experiments include all genes known to
modulate non-specific and specific host defence mechanisms,
i.e., genes encoding cytokines (58), major histocompadbility
complex (MHC) proteins ( 3 , 3 9 , 5 0 , 9 4 ) , T-cell receptors
(TCRs) (76) and proteins conferring specific disease resistance
(1, 4 0 , 8 6 ) . Transgenes may comprise naturally occurring
resistance genes or genes which enhance the immune
response, or they may consist of in vitro designed gene
products (e.g., antisense RNA, DNA vaccines). The different
strategies of transgene interference with the pathogen-host
interaction are listed in Table II. Referring to the site and
mode of action and the source of the effective agent, some of
the strategies are termed 'intracellular', 'extracellular', 'genetic'
and 'congenital' immunisation (64). The authors discuss the
differing transgenic strategies according to the gene transfer
method and the immunisation approach.
Somatic gene transfer
Genetic immunisation (DNA vaccination)
The stimulation of an antibody response by delivering
plasmids coated onto gold beads driving the expression of a
foreign protein in mice was first described in 1992 (90). The
Table II
Transgenes and transgenic concepts aiming at the increase of disease resistance
Transgene target
Transgenic strategy
Examples of transgenes encoding
Pathogen attachment and penetration into host cells, tissues or organs
Extracellular immunisation
Intracellular Immunisation
Congenital immunisation
Genetic immunisation
Pathogen replication and progeny release
Intracellular immunisation
Soluble pathogen receptors
Mutated cellular pathogen receptors
Substances interfering with pathogen entry
Antibodies
Antigens
Antisense RNA
Ribozymes
Intrabodies
Inhibitors of replication
Dominant negative mutant pathogen proteins
competing with the wild-type proteins
Latency or spread of the pathogen through the host organism
Extracellular immunisation
Congenital immunisation
Genetic immunisation
Products inducing or maintaining latency
Antibodies
Antigens
Antimicrobial agents
Host Immune system
Intracellular Immunisation
Extracellular immunisation
Cytokines
Cytokine receptors
Intracellular effectors of cytokine signalling
Identified host susceptibility genes
Gene targeting (knockout/
gene replacement!
Gene targeting constructs
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Rev. sci. tech. Off. int. Epiz., 17 (1)
authors choose the term 'genetic immunisation' to describe
the somatic DNA delivery and utilisation of antigen-encoding
gene constructs rather than antigens per se for vaccination.
DNA constructs have been delivered to individuals by several
routes, including intramuscular, intravenous, intradermal
and needleless (jet) injections and micro-bombardment.
Depending on the method of administration, the foreign DNA
was expressed locally or systemically to levels high enough to
induce specific antibody production ( 2 3 , 2 8 , 2 9 , 9 2 , 9 3 ) . In
addition to the use of naked DNA or bound DNA, nucleic acid
vaccines may be targeted to specific tissues, such as mucosal
sites, for the induction of local immune responses. With
respect to this technique, Sizemore et al. have generated a
bacterial vector capable of DNA delivery ( 8 2 ) . Mice
immunised with recombinant Shigella containing plasmid
expression constructs produced cellular immune responses
and high titres of specific antibodies. This strategy opens up
the possibility for the oral delivery of functional DNA with the
potential to induce a local or systemic immune response.
Tsukamoto et al. have demonstrated that a single intravenous
injection of DNA/lipopolyamine complexes into pregnant
mice resulted in successful gene expression in the embryos
and neonates (91). This opens new aspects for basic research
and practical use in neonate vaccination. DNA vaccines have
the great advantage of relatively short developmental times,
ease of large-scale production and cost-effectiveness in
development, manufacture, storage and distribution. DNA
vaccine technology also simplifies the process of vaccine
antigen discovery. Mixtures of expression plasmids containing
fragments of the genome of a given pathogen can be tested for
protective efficacy. Using this approach of 'expression library
immunisation' (ELI) (6), it should be possible to successively
fractionate and test the DNA mixtures and identify the
plasmid(s) encoding the protective antigens.
In veterinary medicine, routine vaccination is mostly applied
to large populations. Methods for reducing costs and
increasing the efficiency of DNA vaccination include jet
injection, gene gun or aerosol application of naked or
complexed DNA ( 9 5 ) . In vivo gene transfer to the airway
epithelium was successfully carried out after intratracheal
administration in mice (91). Prior to use in farm animals, such
delivery methods have to be tested in these species and the
necessary
safety
aspects
(biodistribution,
long-term
expression, DNA persistence, germ-line integration) have to
be considered. Currently, non-viral DNA delivery methods
are preferred to the viral-based DNA vector systems for safety
reasons. The use of viral vectors for immunisation in
veterinary medicine is discussed elsewhere ( 1 0 5 ) .
Immunomodulatory molecules, such as cytokines or
costimulatory molecules, may be effective at enhancing
immune responses against antigens expressed by genetic
immunisation. These molecules could be delivered as
recombinant polypeptides or could be expressed in situ using
plasmid DNA (106). The somatic gene transfer of cytokine
transgenes alone might also be used for the enhancement of
disease resistance, as discussed below under 'extracellular
immunisation'.
Germ-line gene transfer
Examples of attempts to introduce disease resistance traits
into mammals by germ-line gene transfer are summarised in
Table III and described briefly below.
Table III
Transgenic animals carrying disease resistance enhancing gene constructs
Transgenic strategy
Gene construct encoding
Tissue-specific
expression
Transgenic farm
animal or mouse
Congenital immunisation
Monoclonal antibodies
Blood
Rabbit, sheep, pig
Intracellular immunisation
Antisense RNA-adenovims
Antisense RNA-bovine
leukaemia virus
Dominant-negative virus protein
Mx proteins
Nrampl
-
Rabbit
-
Sheep
Pig
Mouse
Bovine TAP
Mammary gland
Mouse
Synthetic cecropin
Blood, lymph
Mouse
Human CRP, rabbit CRP
Liver and other organs
Mouse
Human lactoferrin
Human lactoferrin
Mammary gland
Cattle
Mouse
Extracellular immunisation
CRP
TAP
Nramp
: C-reactive protein
: tracheal antimicrobial peptide
: natural resistance-associated macrophage protein
—
Biological effect
Antigen binding; aberrant
immunoglobulins
Enhanced resistance to challenge
with virus in vitro and in vivo
Not recorded
Not detected
Enhanced resistance to challenge
with bacteria
Antimicrobial activity of purified
active protein
Enhanced resistance to challenge
with bacteria
Enhanced resistance to challenge
with bacteria
Not recorded
Purified active protein identical to
wild-type peptide
References
(55,98)
(32,48)
(25,26)
(62)
(38)
(104)
(74)
(89,103)
(49)
(67,71)
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Rev. sci. tech. Off. int. Epiz., 17 (1)
Deletive gene transfer
Knockout or replacement of susceptibility or disease
genes
The technique of altering the genome by homologous
recombination ( 6 9 ) is made possible by the availability of
totipotential cells in farm animals or by using the nuclear
transfer cloning technique. This allows the targeted disruption
of disease causing genes or the replacement of mutant alleles
by alleles causing enhanced resistance. A widely discussed
example in this context is the generation of prion-resistant
flocks and herds. Transmissible spongiform encephalopathies
(TSEs) are thought to be caused by a malfolded version of the
normal cellular prion protein (2, 7 3 , 1 0 0 ) . The favoured
'protein-only' hypothesis maintains that the prion is devoid of
nucleic acids, although the participation of additional
infectious agents (51) or the 'unconventional virus' hypothesis
cannot be ruled out completely. The prion diseases occur in
humans (e.g., Creutzfeldt-Jakob disease [CJD]) and animals,
including sheep (scrapie) and cattle (bovine spongiform
encephalopathy [BSE]). There is now considerable concern
that BSE may be transmissible to humans, resulting in a new
variant of CJD (19, 44). The observation that mice lacking the
endogenous prion protein are resistant to prion disease ( 1 0 0 )
suggested the generation of prion gene-free sheep and cattle
by gene targeting. The availability of the appropriate
techniques in sheep and cattle will allow the establishment of
proof of principle. However, the production of prion-free
farm animals is less practical, since it would be an enormous
task to replace the many differing breeds and to preserve the
genetic variety. In addition, such animals might suffer some
deleterious effect from the depletion of the prion gene, as has
been observed in mice (2, 7 3 , 9 9 ) . However, prion-diseaseresistant farm animals would undoubtedly be of benefit for
gene farming purposes. For domestic animals, a more efficient
approach might be to introduce prion transgenes known to
act as dominant-negatives or to replace the existing prion gene
alleles by prion gene variants resistant to the development of
TSEs.
traits such as germ-line-encoded immunity into farm animals.
Whether or not the efforts required for optimising the concept
of 'congenital immunisation' are justified by its benefits in
terms of increased disease resistance in a certain species
remains to be investigated. Following this route, one has also
to keep in mind that the high mutation rates of most
infectious pathogens will readily create novel subtypes able to
escape the immunity of the transgenic animal by changing
their antigenic determinants. In the transgenic animals
generated by Weidle et al. ( 9 8 ) , antibodies were not only
detected in the blood stream but also in the colostral milk
(65). In this experiment, the heavy and light chain
monoclonal antibody encoding regions were driven by the
endogenous mouse immunoglobulin promoters. The
expression of antibodies can be more efficiently directed to
the mammary gland using mammary gland-specific promoter
regions. Milk containing recombinant antibodies would be of
high value for the prevention of neonatal diseases in suckling
offspring. In addition, this technique is used for the
production of monoclonal antibodies for therapeutic or
diagnostic use. It is also worth mentioning that the antibody
expression technology is applicable to antigens that are
non-proteinous.
Intracellular immunisation
The term 'intracellular immunisation' was originally proposed
for the overexpression in the host of an a b r r a n t form
(dominant-negative mutant) of a viral protein that is able to
interfere strongly with cellular entry or the replication of the
wild-type virus ( 5 ) . This definition was then extended to
include all approaches based on intracellular expression of
transgene products which inhibit the multiplication of
pathogens in host organisms. Thus different strategies using
intracellular antibodies (intrabodies), antisense RNAs and
ribozymes,
dominant-negative pathogen proteins and
host-derived antipathogenic proteins are summarised by this
term.
Intrabodies
Additive gene transfer
Congenital immunisation
'Congenital immunisation' is defined as transgenic expression
and germ-line transmission of a gene encoding an
immunoglobulin specific for a pathogen. The animal therefore
shows congenital immunity without prior exposure to that
pathogen ( 6 5 ) . As shown by many investigations, cloned
genes coding for monoclonal antibodies can be expressed in
large amounts in transgenic mice. The approach was tested in
farm animals by expressing the gene constructs encoding
mouse monoclonal antibodies in transgenic rabbits, pigs and
sheep ( 5 5 , 9 8 ) . Both experiments resulted in transgene
expression but also revealed some unexpected findings, e.g.,
aberrant sizes of the transgenic antibody or little antigenbinding capacity. These results were explained by
heterologous immunoglobulin chain associations and/or
deviant post-translational modifications. Nevertheless, both
experiments illustrate the potential of introducing beneficial
Recent advances in recombinant antibody technology have
allowed the genes encoding antibodies to be modified so that
the antigen-binding domain is expressed intracellularly ( 5 1 ) .
This is achieved by mutation or deletion of the hydrophobic
leader sequence for secretion of the recombinant antibodies.
Intracellularly, the antibodies can be directed to the desired
subcellular compartment by adding the appropriate
localisation signal (11). In the whole animal, the expression of
an intrabody can be restricted temporally or spatially by using
a promoter that is either tissue- (cell-) specific, active at only a
certain time in development or upon stimulation, or both.
Intrabodies represent a versatile tool to modulate the function
of selected intracellular gene products
('phenotypic
knockout') but also promise to be powerful in the defence
against infectious pathogens ( 1 0 , 6 0 ) . The recombinant
antibodies most often utilised for this approach have been
single-chain variable region fragments (scFv), but other
formats (whole antibodies, antigen-binding fragments) have
Hev. sci. tech. Off. int. Epiz., 17 (1)
370
been used as well. Single-chain Fv proteins are recombinant
antibodies derived from monoclonals and are able to bind to
their antigens with affinities similar to those of the parental
antibodies (37). They consist of only the variable L chain and
the variable H chain domains covalently linked by an
engineered polypeptide linker. Due to their small size, lack of
assembling requirements and easy derivation, these proteins
are well suited for the design of new therapeutics and
transgenic approaches. At present, intrabodies are mainly
investigated for their potential in human gene therapy. In
agriculture, the approach has been successfully tested in
plants (65), but awaits establishment in transgenic farm
animals.
Antisense
ribonucleic
acid and
ribozymes
Antisense
nucleic acids
comprise
short
synthetic
oligonucleotides (often oligodesoxyribonucleotides) and
substantially longer sequences formed by PJMA transcribed
either in vitro or in vivo. Antisense molecules can inhibit the
complementary messenger RNA (mRNA) at multiple levels in
the expression and replication of a gene: for example, by
blocking the mRNA translation; by increasing the sensitivity
of mRNA to cellular double-stranded RNA ribonucleases; by
blocking the RNA expon from the nucleus; and by base
modifications of the RNA. Ribozymes, like antisense RNA, are
RNA molecules that bind to their complementary sequences.
However, they function additionally to cleave the target RNA
(22, 56). Antisense and ribozyme constructs have been tested
successfully in transgenic animals (83). Antisense technology
was applied in mice and rabbits to prevent infectious diseases.
Transgenic mice expressing antisense RNA targeted to the
retroviral packaging sequences of Moloney murine leukaemia
virus did not develop leukaemia following challenge with
infectious viruses ( 4 2 ) . In another report, the nucleocapsid
protein gene of the mouse hepatitis virus was the target of
inhibition by antisense RNA. The nucleocapsid protein is
crucial for the packaging process of the viral genome. Mice
expressing the antisense gene construct were shown to be
more resistant to infection leading to death than nontransgenic littermates ( 7 9 ) . Ernst et al. described the
generation of transgenic rabbits expressing an antisense
construct complementary to adenovirus h5 RNA ( 3 2 ) .
Primary cells from transgenic animals were found to be 9 0 %
to 9 8 % more resistant to adenovirus infection than cells from
control animals. The effect of an antisense RNA transgene
targeted against the 5'-untranslated long-terminal repeat of
the bovine leukaemia virus (BLV) on the virus replication and
spread in a rabbit model system was studied ( 4 8 ) . BLV, a
retrovirus, causes persistent lymphocytosis and B-lymphocyte
lymphoma in cattle and sheep, but is also virulent in
laboratory animals. Transgenic rabbits showed enhanced
resistance to BLV infection compared to wild-type animals.
Although the transgenics also produced antibodies to viral
proteins, the haematological changes observed in wild-type
rabbits were considerably less pronounced in these animals.
The use of antisense RNAs as anti-pathogenic agents has
several limitations which are mainly imposed by the
stoichiometric nature of the inhibition. Therefore, ribozymes
offer the possibility to develop antisense constructs that not
only result in RNA-RNA hybrids, but in addition catalytically
cleave a phosphodiester bond in the target RNA strand. The
catalytic RNA (i.e., the ribozyme) would not be consumed
during the cleavage reaction, thus a large number of substrate
molecules could be processed ( 7 8 ) . So far, there are no
ribozyme transgenic animal models reported to show
resistance to infectious diseases. A hammerhead ribozyme
flanked by antisense sequences directed against regulatory
proteins of BLV was shown to inhibit BLV expression in
persistently infected cells (21). Taken together with the in vivo
data obtained from transgenic rabbits ( 4 8 ) , this suggests the
possibility of generating transgenic ruminants that will be
resistant to BLV-induced diseases.
The creation of animals containing intrabodies, or antisense or
ribozyme constructs, against common pathogens is used in
cases where the disease-causing organisms are well known.
Intrabodies or anti-RNA agents could be raised against
conserved epitopes of an essential protein or early RNAs of the
pathogen. A more effective strategy would be to provide low
levels of transgene products against multiple target proteins or
RNAs in the disease-causing organism, thereby reducing the
chance that the pathogen would escape by mutagenesis.
Dominant-negative
mutant
pathogen
proteins
The original concept of intracellular immunisation aims at the
inhibition of viral multiplication by interference of mutant
and wild-type virus proteins. The extended definition
includes all approaches using (intra)cellular antiviral agents
(35). Clements et al. generated transgenic sheep expressing
the maedi-visna virus envelope (E) gene ( 2 5 ) . Maedi-visna
virus belongs to a subfamily of ovine retroviruses that cause
encephalitis, pneumonia and arthritis in sheep. Its
E glycoprotein is responsible for binding the virus to host
cells. The target cell for maedi-visna virus replication in
infected sheep is the monocyte/macrophage. Maedi-visnainfected cells express E protein on their surface which causes
immune responses to the virus. Expression of a gene construct
consisting of the maedi-visna 3'-untranslated enhancer
region fused to the E gene in transgenic sheep had no obvious
deleterious effect. Transgene expression was detectable
predominantly in macrophages and to a smaller extend in
fibroblasts and lymphocytes. Thus, the generated transgenic
sheep lines provide a model for studying the potential of a
retroviral E glycoprotein to prevent infection and/or to
modulate disease in its natural host after virus challenge (26).
Specific
disease
resistance
genes
This term refers to single genetic loci responsible for defined
disease resistance traits. Unfortunately there are only rare
cases of one gene causing a certain resistance described.
Examples are provided by the Mx sytem ( 4 0 ) , the natural
Hev. sci. tech. Off. int. Epiz., 17 (1)
resistance-associated macrophage protein (Nrampl) gene (1)
or the retroviral (Friend virus) susceptibility/resistance genes
in mice (86).
The Mx system was originally characterised in mice and is
known to be able to block the multiplication of certain
negative-stranded RNA viruses. The generation of Mx
transgenic mice is reviewed elsewhere (4, 40). The attempt to
introduce resistance to influenza A viruses into pigs by
transferring mouse M x l gene constructs into pigs failed.
Extensive protein analysis did not detect mouse M x l protein
in transgenic pigs ( 6 2 ) . The underlying reasons may be
transgene rearrangement or, more likely, the possibility that
the mouse M x l promoter does not function optimally in pig
cells.
The mouse Nrampl gene (formerly the Bcg/lty/Lsh locus)
determines the susceptibility of inbred strains to infection
with unrelated intracellular parasites. In livestock species, the
locus was also found to be associated with intracellular
parasite resistance ( 3 3 , 4 6 ) . In mouse inbred strains, the
susceptibility to infection is associated with a single amino
acid substitution of the protein. Gene transfer of the
non-mutated Nrampl gene into susceptible mice conferred
resistance to infection with Mycobacterium
bovis and
Salmonella Typhimurium (38).
Genetic resistance to certain retroviruses has been observed as
a polymorphic trait in several experimental species. One of
the identified loci in mice, Fv-4, resembles the 3 ' half of a
murine leukaemia virus genome. Expression of the mouse
Fv-4 encoding only the viral envelope protein in transgenic
mice conferred resistance to infection with ecotropic
retroviruses ( 5 3 , 5 4 ) . The mechanism of Fv-4 resistance is
thought to be related to the phenomenon of viral interference,
i.e., competition of the synthesised envelope protein with
exogenous virus for the virus receptor. Although the
resistance is mediated by an endogenous mouse gene, the
strategy
resembles
the
concept
of
overexpressing
dominant-negative pathogen proteins.
Extracellular
immunisation
A variety of strategies for the enhancement of disease
resistance by transgenesis use gene products which exhibit
their antipathogenic activity extracellularly. According to the
immunisation strategies described above, the authors refer to
methods described next as 'extracellular' immunisation.
Cytokines are potent regulators of the innate and acquired
immune response ( 5 8 ) . As cytokines form a complex
interacting network which regulates cellular growth and
differentiation in physiological and pathophysiological states,
experiments involving the transfer of cytokine genes have to
be carefully designed. Aberrant expression of any cytokine
may result not only in the desired positive immune-regulatory
effects but also in no effects or in deleterious 'side-effects', as
has been experienced in therapeutical uses of cytokines and
371
after deviant expression of cytokines in a variety of transgenic
mouse lines (63). Recent progress in understanding the signal
transduction pathways and transcription factors activated by
cytokines ( 1 8 , 6 8 ) promises to open up new therapeutic
approaches as well as novel strategies of gene transfer
experiments which will aim at the improvement of the
immune response (i.e., the genetic modification of distinct
'cytokine-specific' signalling components) rather than the
transfer of cytokine encoding genes per se. In contrast to the
germ-line integration of cytokine gene constructs, the
transient expression of cytokines by somatic gene transfer has
been reported to show the required effects (43, 8 7 ) .
Peptide-based antimicrobial defence is an evolutionary
ancient mechanism of host response found in a wide range of
animals (from insects to mammals) and plants (12, 101).
These peptides have been classified in four classes based on
structural features: the magainins, defensins, melittins and
cecropins. These small lytic peptides interact with lipid bilayer
membranes to cause osmotic disruption and cell death.
Bacterial, protozoan, fungal and damaged eukaryotic cells are
most susceptible to disruption. These peptide antibodies are
currently thought to solve some of the problems encountered
with multidrug-resistant microbes. Recently two transgenic
approaches used constructs encoding antimicrobial peptides.
Tracheal antimicrobial peptide (TAP) is a member of the
|3-defensin family which is isolated from the tracheal mucosa
of the cow, and exhibits broad-spectrum activity when
assayed in vitro against several microbes, including respiratory
pathogens. The bovine TAP complementary DNA (cDNA)
controlled by the mammary gland-specific WAP (whey acidic
protein) promoter was expressed in mice without malign
phenotype. The uptake of bovine TAP with the milk also
caused no deleterious side-effects in suckling pups. The
recombinant bovine TAP purified from mouse milk showed
correct folding and processing of the peptide antibiotic and
hence antimicrobial activity ( 1 0 4 ) . A similar gene construct
might be used in transgenic farm animals for gene farming
purposes, i.e., the large-scale production of recombinant TAP.
In addition, the mammary gland-specific expression of
antimicrobial peptides could be used to prevent enteric
infections of neonates, e.g., the enteric Escherichia
coli
infection in piglets. In the second experiment, a synthetic
cecropin-class antimicrobial peptide-encoding sequence was
fused to the interleukin (IL) 2 promoter, including the first 2 0
amino acids of IL-2 serving as a signal peptide for secretion.
The expression cassette was chosen to produce the
antimicrobial agent in the blood and the lymph.
Lymphoid-specific transgene RNA expression could be
detected in transgenic mice upon treatment with IL-2
inducers. The antimicrobial activity was tested in a Brucella
abortus challenge of the mice. After four weeks, there were
significantly lower numbers of bacteria in the spleens of
transgenic mice compared to non-transgenic littermates (74).
Although some animals showed low-level constitutive
transgene expression, the transgenic animals attained normal
size, life spans and reproductive performance. The transgenic
372
mice serve as a model for future use of such gene constructs in
gene therapy or the generation of livestock species which are
generally more disease-resistant.
Following an acute phase stimulus, such as infection or
physical injury, many liver-derived plasma proteins are
increased in concentration. These provide enhanced
protection against invading micro-organisms, limit tissue
damage and promote a rapid return to homeostasis.
C-reactive protein (CRP) is one of these acute-phase proteins
(85, 88). Mice expressing high levels of rabbit or human CRP
under the control of constitutively active promoters were
partially protected against lethal challenges by mediators of
septic shock, including bacterial endotoxin, and showed a
significantly increased resistance to infection
with
Streptococcus pneumoniae ( 8 9 , 1 0 3 ) .
Lactoferrin
is an iron-binding
glycoprotein
found
predominantly in milk and granulocytes, and to a lesser extent
in exocrine fluids, such as bile and tears. Despite the fact that
its specific function has yet to be fully elucidated, lactoferrin
has been proposed to play a role in iron uptake by the
intestinal mucosa and to act as a bacteriostatic agent by
withholding iron from iron-dependent bacteria. In addition, it
may be involved in phagocytic killing and immune responses
(57). Transgenic mice were produced carrying either human
lactoferrin cDNA or genomic sequences controlled by the
regulatory regions of the bovine ctSl casein gene. The
recombinant human lactoferrin produced in the milk of the
transgenic mice did not differ from the human milk-derived
protein with respect to biochemical properties and the
immunoreactwity (67, 7 1 ) . The generation of a transgenic
bull carrying a mammary gland-specific human lactoferrin
construct was also reported (49). However, there is no report
on expression studies of transgenic offspring. The
experiments described clearly aim at the production of
nutraceuticals, i.e., 'humanised' milk in transgenic cattle.
Nevertheless, similar constructs might be used to prevent
mastitis or bacterial infections of neonates in farm animals.
Conclusions
Gene transfer technology enables the direct introduction of
novel genes into farm animals. The biological outcome of the
transgene in the host genome can be measured in a few
generations. Breeding success can be achieved in a shorter
time compared to classical breeding programmes. To date,
classical breeding has not yet resulted in significant increase in
resistance to disease. However, gene transfer experiments can
be only carried out in a limited number of animals. Once the
positive biological outcome of the artificially added allele in
the founder animal and its offspring is established, the novel
trait has to be spread in the breeding population by
conventional strategies. Gene transfer programmes which aim
at an improvement in disease resistance are currently
performed at the level of basic research. So far, no successful
Rev. sci. tech. Off. int. Epiz. 17 (1)
resistance-enhancing gene transfer experiment in farm
animals has been reported. A variety of encouraging
experiments in mouse models can be used to define the future
research directions for farm animals. However, the
applicability of each approach which has proved to be
successful in mice must be assessed for the larger mammal
used for production or other purposes.
In general, gene transfer experiments in farm animals face
several obstacles, as follows:
0 the efficiency of transferring genes by microinjection into
pronuclei of zygotes is still too low for routine use in
agriculture
it) DNA microinjection results in random integration of the
gene constructs in the host genome. Thus, the transgene
expression often underlies 'chromosomal position effects',
which results in uncertainty about the expected tissue
specificity and in variations of transgene expression levels in
different transgenic lines carrying identical DNA constructs.
One approach to achieve a strict spatio-temporal pattern of
expression from genes of interest is the use of large gene
constructs which provide extensive sequences flanking the
coding unit of the gene to avoid unwanted side effects of
transgene expression. These constructs could be provided by
the currently expanding artificial chromosome vector
technology (96). Recently, a report of the first group of yeast
artificial chromosome DNA transgenic farm animals (rabbits)
has been published (17)
Hi) although promising alternative gene transfer methods
have been reported, the sperm-mediated transgenesis and
gene transfer into foetal cell lines with subsequent nucleus
transfer have yet to be evaluated for reliability and efficiency
iv) despite the enormous progress made in the identification
of regulatory elements of gene regulation, there is an ongoing
demand for basic and applied research on the control of gene
expression using laboratory and farm animals
y) the biosafety issues arising with the generation of
transgenic farm animals and the food safety evaluation of
genetically engineered nutrients remain to be resolved, mainly
on the level of politics and legislation ( 4 5 , 6 1 ) . The ultimate
success of transgenic animals will depend upon the public
acceptance of the product.
In addition, gene transfer programmes for the generation of
animals carrying disease resistance constructs must consider
carefully the possibility of creating or accumulating
pathogenic agents able to escape the introduced host defence
gene. For example, the strategy of introducing mutated
pathogen genes into animals might result in recombination
events with wild-type pathogens, thus creating resistant
strains or even altered species specificity. A similar scenario
has been observed under experimental conditions in
intracellularly immunised
transgenic plants.
Neither
transgenesis nor conventional prophylactic or therapeutic
measures will be able to overcome the need for optimal
373
Rev. sci. tech. Off. int. Epa., 17(1)
animal husbandry
problems.
condition
to
avoid
disease-related
The
ongoing genome projects in mammals and the
electronically accessible formats of comparative maps will lead
to the identification of novel candidate disease-resistance
genes and of new regulatory elements for gene transfer
experiments ( 3 1 , 6 6 , 9 7 ) . While the creation of transgenic
farm animals offers tremendous promise for enhancing the
quality of production and levels of disease resistance in farm
animals, time, money and public acceptance will determine
the feasibility of such an undertaking.
Les méthodes transgéniques appliquées à l'amélioration
de la résistance aux maladies chez les animaux d'élevage
M . Müller & G. Brem
Résumé
G r â c e a u x t e c h n i q u e s d e la g é n é t i q u e m o l é c u l a i r e e t d e la biologie d e la
r e p r o d u c t i o n , on p e u t t r a n s f é r e r de l'acide d é s o x y r i b o n u c l é i q u e é t r a n g e r à des
m a m m i f è r e s . Le r e c o u r s à de n o u v e l l e s m é t h o d e s v i s a n t à modifier la r é s i s t a n c e
ou la sensibilité a u x m a l a d i e s c h e z les bovins a pour but d ' a m é l i o r e r l ' é c o n o m i e
de
l'élevage
e t le b i e n - ê t r e
d e s animaux.
Les recherches
actuelles sur
l'amélioration d e la r é s i s t a n c e a u x m a l a d i e s par t r a n s f e r t d e g è n e s s ' a r t i c u l e n t
a u t o u r d e s trois p r i n c i p a l e s s t r a t é g i e s s u i v a n t e s :
a) t r a n s f e r t de g è n e s s o m a t i q u e s , c ' e s t - à - d i r e v a c c i n s à a c i d e n u c l é i q u e ;
b) t r a n s f e r t de g è n e s invalidés de la lignée g e r m i n a l e ( g e n e k n o c k o u t ) ;
c) t r a n s f e r t de g è n e s d e la l i g n é e g e r m i n a l e a v e c e f f e t d'addition.
Ces
stratégies
visent
soit
à l'expression,
provisoire
ou permanente, de
c o m p o s a n t s c o n n u s p o u r leur a c t i o n s u r l e s m é c a n i s m e s
immunologiques,
s p é c i f i q u e s e t non s p é c i f i q u e s , de l'hôte, soit à l'invalidation de g è n e s d o n t on sait
qu'ils sont à l'origine de la sensibilité à u n e m a l a d i e . S e l o n l'origine du g è n e actif,
du site ou du m o d e d ' a c t i o n , on parle de s t r a t é g i e s d ' i m m u n i s a t i o n « g é n é t i q u e »,
« c o n g é n i t a l e », « i n t r a c e l l u l a i r e » o u « e x t r a c e l l u l a i r e ». Les c o n s é q u e n c e s d e
c h a q u e e x p é r i e n c e de t r a n s f e r t d e g è n e s d o i v e n t ê t r e s o i g n e u s e m e n t é v a l u é e s ,
n o t a m m e n t e n c e qui c o n c e r n e l'apparition possible d e n o u v e l l e s r é s i s t a n c e s à
d e s a g e n t s p a t h o g è n e s , la disparition des b a r r i è r e s d ' e s p è c e s ou e n c o r e la p e r t e
d e s restrictions d e s t y p e s c e l l u l a i r e s .
Mots-clés
Constructions de gènes - Gain de fonction - Immunisation - Perte de fonction Résistance aux maladies - Transgène - Vaccin à A D N .
Rev. sci. tech. Off. int. Epiz., 17 (1)
374
Métodos transgénicos para incrementar la resistencia
a la enfermedad en animales de granja
M . Müller & G. Brem
Resumen
Existen t é c n i c a s d e g e n é t i c a m o l e c u l a r y d e biología d e la r e p r o d u c c i ó n q u e
p e r m i t e n t r a n s f e r i r A D N a j e n o a u n m a m í f e r o . C o n s i d e r a c i o n e s d e índole t a n t o
e c o n ó m i c a c o m o d e b i e n e s t a r a n i m a l j u s t i f i c a n la a d o p c i ó n d e s i s t e m a s
n o v e d o s o s p a r a m o d i f i c a r la r e s i s t e n c i a o la s u s c e p t i b i l i d a d d e l g a n a d o a las
e n f e r m e d a d e s . Tres s o n l a s e s t r a t e g i a s b á s i c a s q u e sigue a c t u a l m e n t e la
i n v e s t i g a c i ó n e n c a m i n a d a a m e j o r a r la r e s i s t e n c i a a la e n f e r m e d a d p o r
t r a n s f e r e n c i a g é n i c a . S e t r a t a d e las s i g u i e n t e s :
a) t r a n s f e r e n c i a d e g e n e s a c é l u l a s s o m á t i c a s , esto e s , v a c u n a s c o n á c i d o s
nucleicos;
b) t r a n s f e r e n c i a s u p r e s o r a d e g e n e s a c é l u l a s d e la l í n e a g e r m i n a l , e s decir,
s u p r e s i ó n e s p e c í f i c a d e g e n e s (gene
knockout);
c) t r a n s f e r e n c i a aditiva de g e n e s a c é l u l a s d e la línea g e r m i n a l .
Estas e s t r a t e g i a s p u e d e n e s t a r o r i e n t a d a s : bien a la e x p r e s i ó n t r a n s i t o r i a o
e s t a b l e d e m o l é c u l a s c o n u n a influencia c o n o c i d a s o b r e l o s m e c a n i s m o s d e
d e f e n s a - e s p e c í f i c o s o i n e s p e c í f i c o s - del h u é s p e d ; o bien a la d i s r u p c i ó n d e
g e n e s c o n u n a f u n c i ó n c o n o c i d a c a u s a n t e d e s u s c e p t i b i l i d a d a la e n f e r m e d a d . En
f u n c i ó n d e l o r i g e n d e l a g e n t e e f e c t o r y d e s u sitio y m o d o d e a c c i ó n , e s a s
e s t r a t e g i a s r e c i b e n el n o m b r e d e i n m u n i z a c i ó n « g e n é t i c a » , « c o n g é n i t a » ,
« i n t r a c e l u l a r » o « e x t r a c e l u l a r » . P a r a evitar q u e u n e x p e r i m e n t o d e t r a n s f e r e n c i a
g é n i c a d é origen a la a p a r i c i ó n d e n u e v a s r e s i s t e n c i a s e n un a g e n t e p a t ó g e n o , a
la abolición d e las b a r r e r a s entre e s p e c i e s o a la s u p r e s i ó n d e las r e s t r i c c i o n e s
i n h e r e n t e s a c a d a tipo celular, e s p r e c i s o e v a l u a r c o n s u m o c u i d a d o los posibles
efectos de cada experimento.
Palabras clave
Adquisición de función - Construcciones génicas - Inmunización - Pérdida de función Resistencia a la enfermedad - Transgen - Vacuna de A D N .
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