Download Genome Modification for Meat Science: Techniques and Applications

BIOTECHNOLOGY
Genome Modification for Meat Science:
Techniques and Applications
Kevin D. Wells
Introduction
Genome modification has the potential to positively impact nearly every aspect of animal agriculture. Until recently,
practical technologies to accomplish sophisticated genome
modification have been successful only in the mouse. However, with the birth of the sheep named Dolly (Wilmut et al.,
1997), the first mammal produced by somatic cell nuclear
transfer, it was illustrated that range of genome modifications
that can be performed in mice may also be accomplished in
livestock. The intent of this paper is to describe the current
state and power of gene transfer and other genome modifications in livestock, primarily mammals. Included are descriptions of the primary techniques, the variety of genetic enhancements that are possible, and a sample of potential and
demonstrated applications genome modification for the meat
animal industry. In this paper, only permanent germline modifications will be considered. There will be no discussion of
transient or in vivo somatic cell transfer of genes (gene therapy).
Genome modification is a phrase used to describe any alteration to a genome including addition of genes otherwise
not found in the species of interest, addition of more copies
of an endogenous gene, gene modification, or gene deletion.
Gene additions are generally called transgenes and gene
modifications or deletions are generally called targeted gene.
It is important to establish the basic aim of genome modification. In scientific forums, popular press, electronic media, and
around dinner tables, gene transfer discussions revolve around
genes. On one level that fact is perfectly appropriate. Gene
transfer, genome modification, transgene, gene knockout—
all of these terms have “gene” at their root. However, the gene
per se is not of great importance to any measurable phenotype or characteristic of food animals. The important factor is
the gene product—the structural protein, the enzyme activity,
the developmental signal, the growth factor, etc. The level,
potency, half-life, time of expression and interactions of the
K. D. Wells
USDA-ARS
Beltsville Area Research Center
BARC-East
Bldg. 200, Room 8
Beltsville, MD 20705
[email protected]
53rd Annual Reciprocal Meat Conference
gene product(s) are the factors that effect economically important traits. For example, transgenic pigs have been produced (Pursel et al., 1998) with the coding region of human
prepro insulin-like growth factor I (IGF-I). The final product of
the transgene is IGF-I. Since the mature protein is identical
between human and pig, a species designation for the gene
does not fully describe the situation. In addition, to say that
the transgene is from the human genome would also be incomplete. The majority of the transgene in this case is the
regulatory elements from chicken skeletal alpha-actin. So, most
of the transgene is from the chicken genome, some of it is
from the human genome, and the final processed product is
identical to pig IGF-I. Since one cannot differentiate human
and porcine IGF-I due to identical amino acid sequences,
should we be concerned that the coding region originated
from an anonymous human? The point of this illustration is to
demonstrate that source DNA and its presence in the genome
is much less important than the product for which it encodes.
And for all genome modifications, the gene product produces
the phenotype. Table 1 illustrates the differences in one carcass characteristic, loin-eye area, between the three classes
of animals produced from the transgenic project above—nontransgenic, non-expressing transgenic, and expressing
transgenic.
What Types of Genome Modifications
Can Be Made?
Two major types of genetic modifications are possible. These
modifications include introduction of a new or redundant gene
and modification of an endogenous gene. In nature these occur by horizontal gene transfer (usually involving virus or bacteria) or by random nucleotide changes (replication/repair
errors or environmental DNA damage). Under laboratory conditions, strategies can be developed to increase the frequency
or specificity of these events as well as to isolate and/or identify extremely rare events. By making or isolating valuable
genome modifications, one can accelerate the rate of genetic
improvement. With the completion of various genome sequencing projects and progress in mapping of genes for quantitative traits, much of the work historically performed by quantitative geneticists may move to the realm of genetic engineers.
Superior alleles found in one breed could be moved to another breed without the numerous back-crosses required to
recover the original phenotype. For example, identification
of a rare allele for disease resistance in a dairy breed could be
87
TABLE 1. Expression of IGF-I transgene is required for
increased loin-eye area.
Transgene Status
Loin-eye
(cm2)a
Standard
Error
Percent
Change
Non-transgenic
Transgenic
(Non-expressing)
Transgenic
(expressing)
33.0
32.4
±1.2
±2.0
0.0
2.0
38.0
±1.2
15.2
Expression of transgene increased loin-eye area, least squares
means, P < 0.0002.
a
moved in a single generation into a beef breed without dilution of the “beef genetics.” Traditional selective breeding could
eventually reach the same goal. Genetic engineering simply
changes the time required. However, other genome modifications are not possible through traditional selection. Only
genetic engineering allows that same gene or allele to be
moved into another species. The sort of bovine genes responsible for a generic resistance to viral infection such as an interferon allele for example would probably be useful in swine
and poultry in addition to cattle. Such genes have been tested
in transgenic mice (Arnheiter et al., 1990; Thimme et al., 1995;
Haller et al., 1995). Though genetic engineering will eventually provide an alternative to traditional selective breeding in
specific circumstances, introduction of new genes and unique
changes to endogenous genes will be the more valued role of
genetic engineering.
Random Integration
Any DNA introduced into the germline of an organism by
means other than normal reproduction can be considered a
transgene. Transgenes can therefore originate from the host
species and thus represent additional copies of an endogenous
gene. In addition, transgenes can be homologues from a different species. They can even be from unrelated species, entirely synthetic, or modified versions of genes found in the
host species.
Often, the goal of a genome modification is to obtain a
somewhat different pattern or level of gene expression than
the endogenous gene can provide. To accomplish this, previously characterized regulatory sequences that are known to
provide the expression pattern desired can be combined with
the coding region of the gene of interest. In this strategy as an
example, one could create a transgene that expressed additional growth hormone from the liver in response to a reduction in pituitary growth hormone. This strategy has been used
to express growth hormone throughout the year instead of
seasonally in transgenic fish (Du et al., 1992).
The process of random integration can be used to provide
alternative expression patterns of endogenous genes or their
homologues as noted above. While useful for modification of
endogenous functions, this strategy can also be used to intro88
duce entirely new capabilities into cells, tissues, or organs.
For example, transgenic mice have been produced that express a bacterial enzyme (lysostaphin) that degrades the cell
wall of Staphylococcus species (D. Kerr, personal communication). There is no mammalian counterpart to this enzymatic
activity. In this situation, the enzyme activity was targeted to
milk by combining the bacterial coding region for lysostaphin
with coding region for the secretory signals of human growth
hormone. The coding region for this fusion protein was then
combined with the mammary specific regulatory elements of
beta lactoglobulin to provide mammary specific expression
of the bacterial enzyme that had been modified for secretion
into milk. As a result of introducing this transgene, the
transgenic mice are resistant to experimentally induced S.
aureus mastitis while the control mice are susceptible. S.
aureus is also incapable of growing in the milk from these
animals. A similar strategy is currently being investigated in
transgenic dairy cattle (Wells et al., unpublished). The ramifications of this strategy to the meat industry is discussed later.
Random integration is not limited to single genes. Multiple
transgenes can also be simultaneously introduced into
transgenic organisms. In animals, the transfer of sets of genes
for biosynthetic pathways to provide biosynthesis of essential
amino acids are under investigation. The feasibility of introducing entire biosynthetic pathways has been demonstrated
in transgenic mammalian tissue culture (Rees; Hay, 1995; Rees
et al., 1994; Rees; Hay, 1993), transgenic mice (Ward; Brown,
1998; Bawden et al., 1995), and transgenic plants (Ye et al.,
2000). These strategies allow one to consider the introduction
into an animal product of a nutritional or otherwise beneficial factor that is not encoded by any gene. The summation of
the enzyme activities produce the novel product.
Introduction of new or additional genes is not limited to
gain-of-function phenotypes. Transgenes can also be engineered to provide an inhibitory effect on an endogenous gene
or gene product. By expressing an antisense RNA or ribozyme,
the effective expression of endogenous genes can be reduced
(Sokol et al., 1998; L’Huillier et al., 1996; Luyckx et al., 1999).
Similarly, expression of antagonists can reduce the effective
expression of an endogenous gene. For example, a mutant
human growth hormone has been engineered and expressed
in transgenic mice (Chen et al., 1991). This dominant negative mutant transgene produces a dwarf phenotype due to
competition for growth hormone receptors between the
transgene product and native growth hormone (Sotelo et al.,
1998). Similar strategies can be developed for protein that act
as part of a larger complex by expressing a protein that make
the complex non-functional.
Like traditional genetic selection, gene addition through
random integration can be used to increase or decrease the
level of a native gene product. In contrast to traditional breeding, gene addition also offers the opportunity to add new gene
products or add homologous gene products from a different
species. The technique is very powerful and versatile. However, this technique does allow precise modification of endogenous genes. For that, one needs to be able to make very
specific changes to the sequence of endogenous genes through
homologous recombination.
American Meat Science Association
Homologous Recombination
With homologous recombination, the genetically modified animal does not necessarily contain a transgene. In this
strategy, precise targeted changes can be made to endogenous
genes. Homologous recombination allows one to change an
allele. For example, if laboratory experiments had discovered
amino acid changes to a particular protein that affected the
half-life, activity, or flavor of the protein, those changes need
not occur in nature to be used in animal agriculture. Instead,
specific changes could be made to the coding region for that
protein to provide an allele that expressed the newly discovered alteration. Once enhanced in a founder animal, the allele could then be introduced into commercial populations
by traditional breeding.
With the ability to make precise changes to genes, comes
the opportunity to disrupt or “knock-out” a gene. An example
of a knock-out gene with agricultural relevance is the prion
protein gene. This gene is required for susceptibility to bovine
spongiform encephalopathy (BSE). In mice, disruption of the
prion protein gene has proven the utility of this knock-out to
agriculture (Manson et al., 1994). Through homologous recombination, a marker gene was inserted into the prion protein gene to create a non-functional, disrupted gene. These
animals are incapable of contracting or propagating prions
(Moore; Melton, 1997). Cattle with this characteristic could
be valuable in halting transfer of BSE.
Until recently, it was thought that embryonic stem cells
(ES-cells) were required to accomplish targeted homologous
recombination in livestock. ES-cells can be maintained in
culture in a non-differentiated state. They can be genetically
manipulated, selected, and analyzed without the need to produce transgenic animals. When the desired modification has
been made and confirmed at the laboratory bench, the cells
can be re-introduced into another embryo where they can
contribute to all of the tissues of the body, including the
germline. When the resulting animals reach sexual maturity,
a proportion of their offspring are produced from gametes that
originated from the ES-cells. These animals have the genetic
modification in every cell and transmit that trait in a Mendelian fashion. In addition to maintaining the ability to contribute to the germline, ES-cells also offer the opportunity to observe and select very rare events.
When attempting to target a specific gene, one can expect
about 1 in every 1,000 cells to integrate the transgene into
their genome. Of those 1,000 transgenic cells, one can expect about 1 in 1,000 to integrate in the desired location and
manner. So, a homologous recombination event is a 1 in
1,000,000 cell event. Genetic selection strategies can be devised that enrich for particular integration events. In this way,
a researcher can start with tens of millions of ES-cells and
have a very small population of likely candidate cells to analyze at the end of the procedure. The ability to isolate these
very rare events is a requirement for homologous recombination in livestock. To this date, germline modification via EScells has only been demonstrated in mice. However, the quest
for ES-cells or functionally equivalent primordial germ cells
53rd Annual Reciprocal Meat Conference
continues in livestock (Mueller et al., 1999; Piedrahita et al.,
1998).
Somatic cells can also be cultured under laboratory conditions. The selection schemes that allow enrichment of homologous recombination events in ES-cells can also be used for
somatic cells. With the introduction of Dolly (Wilmut et al.,
1997), the first clone produced from a somatic cell, ES-cells
were illustrated to not be required for homologous recombination in livestock. It is now apparent that transgenic, knockout, and targeted animals can be made via somatic cell cloning. An outline of this procedure is shown in Figure 1.
Techniques
Several techniques exist for transgenic animal production.
Each technique offers different advantages. The difference in
efficiencies, repeatability, and complexity are illustrated in
Figure 2. Each of the major methodologies are discussed below.
Pronuclear Microinjection
Pronuclear microinjection is the standard method of producing transgenic animals when a simple gene addition is
required. Originally described by Gorden et al. (Gordon et
al., 1980) and adapted to livestock by Hammer et al. (Hammer et al., 1985), this procedure involves direct injection of
FIGURE 1.
Somatic Cell Nuclear Transfer
Pipette is Aligned
with Polar Body
and Metaphase Plate
Development Continues Under
Instructions from the New Genes
Polar Body and
Metaphase Chromosomes
Are Captured
Each Cell Retains the
New Genome
All Chromosomes
Are Removed
New Genome is Propagated
Discarded Chromosomes
Are Replaced with a
Genetically Enhanced Cell
New Nucleus is Released
to Replace Genome
An Electrical Pulse Fuses
the Oocyte and New Cell
89
FIGURE 2.
Retrovirus-Mediated
Gene Transfer
Transfer DNA
to Cells
Somatic Cell
Nuclear Transfer
Transfer DNA
to Cells
Pronuclear
Microinjection
Sperm-Mediated
Gene Transfer
Transfer DNA to
Embryo
Transfer DNA to
Sperm
Cells Make Virus
with New DNA
Virus is Transferred
to Oocyte
Remove
Chromosomes
from Oocyte
Fertilize
After Viral Infection,
Sperm are Added to
Fertilize the Oocyte
Nearly 100%
Transgenic
Replace Genome
Exactly 100%
Transgenic
1–30%
Transgenic
0–100%
Transgenic
the transgene into the pronucleus of zygotes. DNA introduced
into the zygote by this method integrates at random. The efficiency of successful integration varies between transgenes,
between experiments, and especially between species. Table
2 illustrates the range of efficiency that can be expected from
microinjection. Most transgenic livestock have been produced
with this procedure.
Somatic Cell Nuclear Transfer
The use of cloning via somatic cell nuclear transfer to
enucleated oocytes is relatively new and is still being developed. However, it is clear that somatic cell nuclear transfer
can be an alternative to pronuclear microinjection in some
species. Offspring have been produced from this procedure
in sheep (Wilmut et al., 1997), cattle, goats (Baguisi et al.,
1999), mice (Wakayama et al., 1998), and pigs (PPL Therapeutics, R. Page, personal communication). Since somatic cell
culture is amenable to both random integration strategies and
homologous recombination strategies, one can expect that
somatic cell nuclear transfer will enable nearly any genome
modification desired. This procedure is much more involved
than pronuclear microinjection. First, the nuclear material of
an unfertilized ovum must be removed. The nucleus of a genetically modified cell can then be used to replace the discarded genome. An intracellular calcium oscillation is induced
in the reconstructed embryo to emulate fertilization so that
development is initiated. Functionally, this procedure produces a zygote that has been constructed from an ovum and
a somatic cell. This procedure is still inefficient and expensive. However, overall efficiencies may be higher for somatic
cell nuclear transfer than for pronuclear microinjection in
some species. Table 3 shows a comparison of the efficiency
of production of transgenic cattle by these two procedures.
Unlike pronuclear microinjection, all animals born from genetically modified somatic cell nuclear transfer are transgenic.
Since the transgene integration step is performed in cultured
cells, the inefficiencies in integration are removed in this strategy. Instead, the reconstruction of embryos and the developmental potential of those embryos represent the majority of
the expense. As Table 3 illustrates, approximately 1/2 the number of oocytes and 1/5 the number of recipients can be required for nuclear transfer as compared to pronuclear microinjection. Though this strategy is sufficiently efficient to be
used to make valuable animals, improvements in the fullgestation development will be necessary for somatic cell
nuclear transfer to become widespread and cost effective for
basic science.
Other Strategies
Two other techniques have been demonstrated for production of transgenic livestock, but have yet been well accepted.
The first of these is viral transfer (Jaenisch; Mintz, 1974;
Soriano; Jaenisch, 1986). This strategy has been further developed with modern viral vehicles to produce transgenic
cattle (Chan et al., 1998). The size of the transgene is limited
TABLE 2. Efficiency of transgenic animal production*.
Animals produced
Transgenic animals produced
Species
No. of
transferred
embryos
No.
% of
Transferred
No.
% of
born
% of
Transferred
Mice
Rabbits
Rats
Cattle
Pigs
Sheep
12,314
1,907
1,403
1,018
19,397
5,242
1,847
218
353
193
1,920
556
15.0
11.4
29.6
19.0
9.9
10.6
321
28
62
7
177
46
17.3
12.8
17.6
3.6
9.2
8.3
2.61
1.47
4.42
0.68
0.91
0.88
*Adapted from Bondioli and Wall, 1998.
90
American Meat Science Association
TABLE 3. Oocytes required to rroduce one transgenic calf.
Somatic-cell Nuclear
Transfera
Oocytes
—
Metaphase II
Manipulated
Enucleated
Fused
Transferred
Blastocysts
Total calves
1073
Pronuclear
Microinjectionb
2022
1618
718
455
411
283
32
1
147
28
Oocytes
Injected
—
—
—
—
Transferred
Blastocysts
Total calves
K.D. Wells, unpublished.
bCalculated from Bondioli and Wall, 1998. Assumed 80%
pronucleous formation rate.
a
by the size of the viral particle, but with appropriately designed constructs, viral transfer could be a valuable, cost-effective tool (Wells et al., 1999).
The second procedure is sperm-mediated gene transfer. Two
modifications of this theme have been successful. The first is
the use of live sperm that have been incubated in DNA are
used to fertilize a normal ovum (Nakanishi; Iritani, 1993;
Lauria; Gandolfi, 1993; Gandolfi, 1998). This strategy appears
to have low repeatability and has therefore not been well accepted. The second variation on this theme involves soaking
dead sperm in DNA and then using the dead sperm for intracytoplasmic sperm injection (ICSI). Thus far, this technique
has only been successfully performed in mice (Perry et al.,
1999).
Opportunities for the Meat Scientist
Genetic Regulatory Elements/Target Tissues
For gene addition experiments, genetic regulatory sequences that will provide the desired expression level and
pattern are required. For the meat industry, transgene expression targeted to muscle will often be the objective. Such regulatory elements have been described and can provide specificity of expression down to the level of specific muscle
fiber-types. For example, the muscle-specific promoter of the
aldolase A gene is expressed mainly in fast-twitch glycolytic
fibers in adult body muscles. Transgenic animals have been
produced which demonstrate that much of this specificity can
be conferred to transgene expression (Spitz et al., 1998). Similarly, regulatory elements from the troponin genes have been
identified that allow transgenes to be slow or fast muscle specific (Nakayama et al., 1996; Corin et al., 1995). Other regulatory elements have been demonstrated to provide transgene
expression preferentially to fast-twitch Type IIB fiber
(Donoghue et al., 1991) and almost exclusively to fast twitch
Types IIA and IIX fibers (Neville et al., 1996). Several promoters have been used for generic skeletal muscle specificity.
53rd Annual Reciprocal Meat Conference
As an example, fast-twitch specific transgene expression
has been used to express myogenin (a muscle-specific transcription factor normally regulated by electrical activity) in
fast-twitch fibers (Hughes et al., 1999). The fast muscles of
such transgenic mice have elevated oxidative mitochondrial
enzymes, reduced glycolytic enzymes, and reduced fiber size.
Much of the current use of the fiber-type-specific regulatory
elements is for basic science questions. However, the tools
are available to begin to enhance muscle characteristics with
specificity to the level of fiber-type.
Phenotypes
Food Safety
As the quality of handling and packaging of meat products
has increased, potential for longer storage time has also increased. Likewise, mechanization has afforded the opportunity to process meat from larger numbers of carcasses. These
factors now interact to provide an opportunity for pathogen
introduction (increased chance of cross contamination) and
longer incubation times for any contaminants. Complete sterilization of meat products may prevent pathogen contaminant that originate from processing plants, but this strategy
poses increased risks “downstream” of the processing plant.
Meat products that are contaminated by the grocer or consumer and then receive a temperature insult may be at risk of
bacterial bloom due the absence of non-pathogenic competition. A strategy to retain sterility or at least reduced bacterial
numbers could circumvent these risks. An animal that produced meat that was inhospitable to microbes could be useful.
In nature, this strategy has been exploited by many organisms. Many frogs for example live in ecosystems with very
high bacterial loads. However, these same frogs are resistant
to bacterial infections. One of the reasons is the presence of
antimicrobial peptides. These small peptides are pore forming molecules that interact with bacterial and fungal cell membranes. If such peptides were expressed in the muscle of
transgenic livestock, one may expect meat products from these
animals would have much reduced risk of allowing bacterial
growth. This strategy would also provide disease resistance
and reduced transmission of zoonotic organisms. This model
has been tested in transgenic mice and does provide resistance to Brucella abortus (Reed et al., 1997). Any peptide that
is gene encoded, non-allergenic, and not harmful to the
transgenic livestock could be used to increase disease resistance, reduce livestock-human disease transmission, and reduce the risk of growth of contaminant bacteria in raw meat
products.
Similar strategies should be explored for reporter genes for
various status issues. For example, could a temperature sensitive reporter gene be used to indicate complete cooking?
Would consumers or processors accept meat that changed
color in response to contamination? Would reporter genes
that could be used to indicate health status of the animal be
useful? In the end, can transgenic animals provide the meat
industry with “built-in” agents to reduce pathogens or report
on various conditions of the muscle or meat? Agents such as
91
pH dependent fluorescence are being developed (Miesenbock;
De Angelis; Rothman, 1998).
Palatability
Though flavor may be important in the end product, and
may even be manipulated via gene transfer, flavor can also be
provided through any of many other strategies. However, tenderness remains to be an issue. If one produced transgenic
animals with a temperature sensitive collagen that depolymerized during cooking, would that animal be of increased
value? Are there other strategies that would be useful—reduced collagen crosslinking in the animal, post mortum protease expression, proteins that affect cold induced muscle
contraction? Gene transfer provides a tool to deliver a protein
to every cell of a tissue. This protein can be intra- or extracellular. As more basic information is discovered on the factors
that affect tenderness, genetic engineering should be considered as a tool to modify the processes governing tenderness.
Nutriceuticals
The next wave of genetically modified crops will likely be
nutriceuticals. That is—the nutritional content of produce or
grain will be increased via transgene products. This can be
accomplished by introduction of novel or additional biosynthetic pathways or by introduction of binding proteins. Would
the meat industry market meat with increased nutritional content? Are there specific nutrients that could induce consumers to increase the consumption of meat products? This strategy does not have to be limited to additions. Expression of
certain fatty acid synthases could be used to change the fatty
acid length or degree of saturation in transgenic animals. Therefore, one could reduce particular fatty acids by increasing
others. This strategy would impact flavor, storage, and perceived healthfulness of meat products. Modification of fat
composition via gene transfer has been successful in oil seed
crops (Knauf; Facciotti, 1995; Dehesh et al., 1996)
egy? Would myostatin, the double muscling gene, be a target
for this strategy? What are the factors currently found in meat
that should be considered for reduction? Likewise, are there
factors that should be considered for increases through gene
transfer? As the science of muscle biology and meat processing proceeds, gene transfer should be considered as a tool for
both investigation and meat modification. The potential of
this technology allows one to take a non-classical point of
view towards not only the problems, but also the definition of
a problem. The character of muscle needs to be considered
not only as a starting material for further processing, but also
as a material that can be manipulated prior to slaughter.
Summary
Until recently, papers such as this one would have offered
futuristic applications of gene transfer that seemed nearly feasible. However, key elements of the proposed strategies would
have likely been still unattainable. In contrast, most applications of gene transfer are feasible in at least some of the livestock species and can now be realized. Several strategies exist for production of transgenic animals. Most rely on the direct
introduction of DNA into embryos. Genes can be added or
removed. They can be modified in expression level or timing.
Novel enzyme activities or end products can be introduced.
Though there are still inefficiencies and costs associated with
transgenic livestock production, the major limitation at this
time is no longer technological. Instead, the major limitation
is imagination and consideration. Have you considered genome modification to help answer your scientific question?
Can genetically enhanced animals provide your stakeholders
with improved, safer, healthier, better quality products? The
genetic engineers cannot sufficiently answer these questions.
Each scientific field must address these issues independently.
However, gene transfer offers a very powerful technology that
has the ability to significantly impact animal agriculture in a
positive way.
Growth
Growth has been a primary target phenotype of transgenic
livestock projects to date (Vize et al., 1988; Pursel et al., 1989;
Pursel et al., 1990; Pursel; Rexroad, 1993). Though this trait
can be significantly impacted by traditional breeding as well
as pharmacologically (Etherton, 1999), it will probably be of
continued interest for genetic engineers. As these projects
develop, one would expect feed efficiency, muscle size, tenderness, color, and other muscle characteristics may also be
affected (Solomon et al., 1994). However, with the refinement
of transgenic strategies to affect growth, specific types of growth
will need to be considered. Are there reasons to increase the
number or size of certain fiber-types? Are there benefits of
reducing certain fiber-types? Since specific fibers can be targeted for transgene expression, more research is needed on
the role of specific fiber-types in economically important traits.
Other Applications
As mentioned above, one can use an antagonistic transgene
to reduce the effective expression of an endogenous gene.
Would leptin, an appetite regulatory, be a target for this strat92
References
Arnheiter, H., S. Skuntz, M. Noteborn, S. Chang, and E. Meier. 1990.
Transgenic mice with intracellular immunity to influenza virus. Cell 62:51–
61.
Baguisi, A., E. Behboodi, D.T. Melican, J.S. Pollock, M.M. Destrempes, C.
Cammuso, J.L. Williams, S.D. Nims, C.A. Porter, P. Midura, M.J. Palacios,
S.L. Ayres, R.S. Denniston, M.L. Hayes, C.A. Ziomek, H.M. Meade, R.A.
Godke, W.G. Gavin, E.W. Overstrom, and Y. Echelard. 1999. Production
of goats by somatic cell nuclear transfer. Nature Biotechnology 17:456–
461.
Bawden, C.S., A.V. Sivaprasad, P.J. Verma, S.K. Walker, and G.E. Rogers.
1995. Expression of bacterial cysteine biosynthesis genes in transgenic
mice and sheep: Toward a new in vivo amino acid biosynthesis pathway
and improved wool growth. Transgenic Research 4:87–104.
Bondioli, K.R. and R.J. Wall. Transgenic livestock. In: Altman, A. (Eds.). Agricultural Biotechnology. New York: Marcel Dekker, Inc., 1998:453–471.
Chan, A.W., E.J. Homan, L.U. Ballou, J.C. Burns, and R.D. Bremel. 1998.
Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proceedings of the National Academy of Sciences, U.S.A. 95:14028–
14033.
Chen, W.Y., M.E. White, T.E. Wagner, and J.J. Kopchick. 1991. Functional
antagonism between endogenous mouse growth hormone (GH) and a
GH analog results in dwarf transgenic mice. Endocrinology 129:1402–
1408.
Corin, S.J., L.K. Levitt, J.V. O’Mahoney, J.E. Joya, E.C. Hardeman, and R.
Wade. 1995. Delineation of a slow-twitch-myofiber-specific transcrip-
American Meat Science Association
tional element by using in vivo somatic gene transfer. Proceedings of the
National Academy of Sciences, U.S.A. 92:6185–6189.
Dehesh, K., A. Jones, D.S. Knutzon, and T.A. Voelker. 1996. Production of
high levels of 8:0 and 10:0 fatty acids in transgenic canola by
overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana.
Plant Journal 9:167–172.
Donoghue, M.J., J.D. Alvarez, J.P. Merlie, and J.R. Sanes. 1991. Fiber typeand position-dependent expression of a myosin light chain- CAT transgene
detected with a novel histochemical stain for CAT. Journal of Cell Biology
115:423–434.
Du, S.J., Z.Y. Gong, G.L. Fletcher, M.A. Shears, M.J. King, D.R. Idler, and C.L.
Hew. 1992. Growth enhancement in transgenic Atlantic salmon by the
use of an “all fish” chimeric growth hormone gene construct. Biotechnology (N.Y). 10:176–181.
Etherton, T.D. 1999. Emerging strategies for enhancing growth: Is there a
biotechnology better than somatotropin? Domestic Animal Endocrinology 17:171–179.
Gandolfi, F. 1998. Spermatozoa, DNA binding and transgenic animals.
Transgenic Research 7:147–155.
Gordon, J.W., G.A. Scangos, D.J. Plotkin, J.A. Barbosa, and F.H. Ruddle.
1980. Genetic transformation of mouse embryos by microinjection of
purified DNA. Proceedings of the National Academy of Sciences, U.S.A.
77:7380–7384.
Haller, O., M. Frese, D. Rost, P.A. Nuttall, and G. Kochs. 1995. Tick-borne
thogoto virus infection in mice is inhibited by the orthomyxovirus resistance gene product Mx1. J. Virology 69:2596–2601.
Hammer, R.E., V.G. Pursel, C.E. Rexroad, Jr., R.J. Wall, D.J. Bolt, K.M. Ebert,
R.D. Palmiter, and R.L. Brinster. 1985. Production of transgenic rabbits,
sheep and pigs by microinjection. Nature 315:680–683.
Hughes, S.M., M.M. Chi, O.H. Lowry, and K. Gundersen. 1999. Myogenin
induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice. Journal of Cell Biology 145:633–642.
Jaenisch, R. and B. Mintz. 1974. Simian virus 40 DNA sequences in DNA of
healthy adult mice derived from preimplantation blastocysts injected with
viral DNA. Proceedings of the National Academy of Sciences, U.S.A.
71:1250–1254.
Knauf, V.C. and D. Facciotti. 1995. Genetic engineering of foods to reduce
the risk of heart disease and cancer. Advances in Experimental Medicine
and Biology 369:221–228.
L’Huillier, P.J., S. Soulier, M.G. Stinnakre, L. Lepourry, S.R. Davis, J.C. Mercier,
and J.L. Vilotte. 1996. Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.
Proceedings of the National Academy of Sciences, U.S.A. 93:6698–6703.
Lauria, A. and F. Gandolfi. 1993. Recent advances in sperm cell mediated
gene transfer. Molecular Reproduction and Development 36:255–257.
Luyckx, V.A., B. Leclercq, L.K. Dowland, and A.S. Yu. 1999. Diet-dependent
hypercalciuria in transgenic mice with reduced CLC5 chloride channel
expression. Proceedings of the National Academy of Sciences,U.S.A.
96:12174–12179.
Manson, J.C., A.R. Clarke, M.L. Hooper, L. Aitchison, I. McConnell, and J.
Hope. 1994. 129/Ola mice carrying a null mutation in PrP that abolishes
mRNA production are developmentally normal. Molecular Neurobiology 8:121–127.
Miesenbock, G., D.A. De Angelis, and J.E. Rothman. 1998. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195.
Moore, R.C. and D.W. Melton. 1997. Transgenic analysis of prion diseases.
Molecular Human Reproduction 3:529–544.
Mueller, S., K. Prelle, N. Rieger, H. Petznek, C. Lassnig, U. Luksch, B. Aigner,
M. Baetscher, E. Wolf, M. Mueller, and G. Brem. 1999. Chimeric pigs
following blastocyst injection of transgenic porcine primordial germ cells.
Molecular Reproduction and Development 54:244–254.
Nakanishi, A. and A. Iritani. 1993. Gene transfer in the chicken by spermmediated methods. Molecular Reproduction and Development 36:258–
261.
Nakayama, M., J. Stauffer, J. Cheng, S. Banerjee-Basu, E. Wawrousek, and A.
Buonanno. 1996. Common core sequences are found in skeletal muscle
slow- and fast-fiber- type-specific regulatory elements. Molecular and
Cellular Biology 16:2408–2417.
Neville, C., D. Gonzales, L. Houghton, M.J. McGrew, and N. Rosenthal.
1996. Modular elements of the MLC 1f/3f locus confer fiber-specific transcription regulation in transgenic mice. Developmental Genetics 19:157–
162.
53rd Annual Reciprocal Meat Conference
Perry, A.C., T. Wakayama, H. Kishikawa, T. Kasai, M. Okabe, Y. Toyoda, and
R. Yanagimachi. 1999. Mammalian transgenesis by intracytoplasmic sperm
injection. Science 284:1180–1183.
Piedrahita, J.A., K. Moore, B. Oetama, C.K. Lee, N. Scales, J. Ramsoondar,
F.W. Bazer, and T. Ott. 1998. Generation of transgenic porcine chimeras
using primordial germ cell-derived colonies. Biology of Reproduction
58:1321–1329.
Pursel, V.G., C.A. Pinkert, K.F. Miller, D.J. Bolt, R.G. Campbell, R.D. Palmiter,
R.L. Brinster, and R.E. Hammer. 1989. Genetic engineering of livestock.
Science 244:1281–1288.
Pursel, V.G., R.E. Hammer, D.J. Bolt, R.D. Palmiter, and R.L. Brinster. 1990.
Integration, expression and germ-line transmission of growth-related genes
in pigs. Journal of Reproduction and Fertility Suppl. 41:77–87.
Pursel, V.G. and C.E. Rexroad, Jr., 1993. Recent progress in the transgenic
modification of swine and sheep. Molecular Reproduction and Development 36:251–254.
Pursel, V.G., K.D. Wells, A.D. Mitchell, T. Elsasser, R.J. Wall, M.B. Solomon,
M.E. Coleman, and R.J. Schwartz. 1998. Expression of IGF-I in Skeletal
Muscle of Transgenic Swine. Journal of Animal Science Suppl. 76:130.
Reed, W.A., P.H. Elzer, F.M. Enright, J.M. Jaynes, J.D. Morrey, and K.L. White.
1997. Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus. Transgenic Research 6:337–347.
Rees, W.D. and S.M. Hay. 1993. The expression of Escherichia coli threonine
synthase and the production of threonine from homoserine in mouse 3T3
cells. Biochemical Journal 291:315–322.
Rees, W.D., S.D. Grant, S.M. Hay, and K.M. Saqib. 1994. Threonine synthesis
from homoserine as a selectable marker in mammalian cells. Biochemical Journal 299:637–644.
Rees, W.D. and S.M. Hay. 1995. The biosynthesis of threonine by mammalian cells: Expression of a complete bacterial biosynthetic pathway in an
animal cell. Biochemical Journal 309:999–1007.
Sokol, D.L., R.J. Passey, A.G. MacKinlay, and J.D. Murray. 1998. Regulation
of CAT protein by ribozyme and antisense mRNA in transgenic mice.
Transgenic Research 7:41–50.
Solomon, M.B., V.G. Pursel, E.W. Paroczay, and D.J. Bolt. 1994. Lipid composition of carcass tissue from transgenic pigs expressing a bovine growth
hormone gene. Journal of Animal Science 72:1242–1246.
Soriano, P. and R. Jaenisch. 1986. Retroviruses as probes for mammalian
development: Allocation of cells to the somatic and germ cell lineages.
Cell 46:19–29.
Sotelo, A.I., A. Bartke, J.J. Kopchick, J.R. Knapp, and D. Turyn. 1998. Growth
hormone (GH) receptors, binding proteins and IGF-I concentrations in
the serum of transgenic mice expressing bovine GH agonist or antagonist. Journal of Endocrinology 158:53–59.
Spitz, F., Z.A. De Vasconcelos, F. Chatelet, J. Demignon, A. Kahn, J.C. Mira,
P. Maire, and D. Daegelen. 1998. Proximal sequences of the aldolase A
fast muscle-specific promoter direct nerve- and activity-dependent expression in transgenic mice. Journal of Biological Chemistry 273:14975–
14981.
Thimme, R., M. Frese, G. Kochs, and O. Haller, 1995. Mx1 but not MxA
confers resistance against tick-borne Dhori virus in mice. Virology 211:296301.
Vize, P.D., A.E. Michalska, R. Ashman, B. Lloyd, B.A. Stone, P. Quinn, J.R.
Wells, and R.F. Seamark. 1988. Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth. Journal of Cell
Science 90:295–300.
Wakayama, T., A.C. Perry, M. Zuccotti, K.R. Johnson, and R. Yanagimachi.
1998. Full-term development of mice from enucleated oocytes injected
with cumulus cell nuclei. Nature 394:369–374.
Ward, K.A. and B.W. Brown. 1998. The production of transgenic domestic
livestock: Successes, failures and the need for nuclear transfer. Reproduction Fertility and Development 10:659–665.
Wells, K.D., K. Moore, and R.J. Wall. 1999. Transgenes go retro. Nature Biotechnology 17:25–26.
Wilmut, I., A.E. Schnieke, J. McWhir, A.J. Kind, and K.H. Campbell. 1997.
Viable offspring derived from fetal and adult mammalian cells [published
erratum appears in Nature 1997 Mar 13;386(6621):200]. Nature 385:810–
813.
Ye, X., S. Al-Babili, A. Kloti, J. Zhang, P. Lucca, P. Beyer, I. Potrykus. 2000.
Engineering the provitamin A (beta-carotene) biosynthetic pathway into
(carotenoid-free) rice endosperm. Science 2000. Jan. 14;287(5451):3035. 287:303–305.
93