GROWTH-RELATED TRANSGENES Early transgenic farm animal research was inspired by the dramatic growth of transgenic mice that expressed a growth hormone GH transgene.[1] A number of trans-
Trang 1Transgenic Animals: Improved Performance
Vernon G Pursel
United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, U.S.A
INTRODUCTION
With the world’s population increasing by more than 70
million people each year, modern agricultural methods
that include animal biotechnology will need to be adopted
if this ever-increasing population is going to avoid
massive conflict over agricultural resources The ability
to isolate, clone, and transfer individual genes into farm
animals provides the opportunity for scientists to produce
transgenic animals with modified traits that are
unattain-able through genetic selection
This article reviews progress on transfer of genes for
productivity traits into farm animals, and some areas that
offer promise for the future
GROWTH-RELATED TRANSGENES
Early transgenic farm animal research was inspired by the
dramatic growth of transgenic mice that expressed a
growth hormone (GH) transgene.[1] A number of
trans-genic pigs and sheep were subsequently produced with
human, bovine, rat, porcine, or ovine GH under the
con-trol of several gene promoters.[2]Although pigs
express-ing GH transgenes grew faster, utilized feed more
efficiently, and were much leaner than their
nontrans-genic siblings, they were not larger and exhibited several
notable health problems, which included lameness,
sus-ceptibility to stress, gastric ulcers, and reproductive
prob-lems.[2]The GH transgenic lambs did not grow faster or
utilize feed more efficiently than control lambs, but they
were much leaner and had serious health problems.[2]
More recently, an insulin-like growth factor-I (IGF-I)
transgene has been used to produce transgenic pigs with
enhanced muscle development and reduced fat in the
carcass, but the transgene did not improve growth rate or
feed efficiency In contrast to the GH transgenic pigs,
definitive phenotypes for the IGF-I transgenic pigs were
not detected, and no gross abnormalities, pathologies, or
health-related problems were encountered.[3]
MODIFICATION OF MILK COMPOSITION
Transfer of genes to alter milk composition has thus far
received little research emphasis, but offers the dairy
industry considerable potential for the future A list of potential changes in milk components worthy of consid-eration is shown in Table 1
About 80% of milk protein from cows is composed of caseins (S1, S2, and k), and whey proteins (b
-lactoglob-ulin, a
-lactalbumin, serum albumin, and g-globulin) make
up the remaining 20%
The caseins form the curds in cheese, whereas the whey proteins represent a less valuable by-product Elimination of b
-lactoglobulin from milk would benefit cheese production because it inhibits rennin’s action on k-casein,[4]and would benefit certain fluid milk consumers because b
-lactoglobulin is responsible for some milk allergies Removal of b
-lactoglobulin from cattle is now technically feasible during transfection of fetal fibroblasts that are then used for nuclear transfer.[7]
While removal of a-lactalbumin (a-lac) from cows’ milk may be beneficial for some consumers, researchers at the University of Illinois have shown that increased concentrations of lactose, which result from a
-lac expression, may be beneficial for piglet growth.[8] They produced transgenic pigs that express bovine a
-lactalbu-min in their milk, which results in a higher milk lactose content in early lactation and a 20 to 50% greater milk yield on days 3 9 of lactation, compared to that of control sows Weight gain of piglets suckling a-lac sows was greater at days 7 and 21 after parturition than that of control piglets Thus, overexpression of a-lac milk protein provides a means for improving growth performance of piglets through enhanced lactation of sows
WOOL PRODUCTION
Three transgenic approaches have been investigated for enhancing wool production or improving wool quality The first involved transfer of bacterial genes that had the capacity to synthesize cysteine from hydrogen sulfide and serine, both of which are available in the rumen Cysteine
is the rate-limiting amino acid for wool production, so an endogenous source of this amino acid has the potential to stimulate wool growth The second approach to improve wool production was to stimulate fiber growth by expression of an IGF-I transgene specifically in wool follicles The third approach was to improve wool fiber quality by altering expression of wool fiber keratin and
DOI: 10.1081/E EAS 120019824
Trang 2keratin-associated protein genes in the wool follicle
cortex.[9] Research on the latter approach is still
under-way in South Australia
ENHANCED ANIMAL HEALTH
Economic losses from diseases of farm animals have been
estimated to amount to 10 to 20% of the total production
costs Use of transgenesis in farm animals holds great
promise for augmenting conventional breeding techniques
to confer animals with improved resistance to these diseases and thereby reduce these losses and enhance animal welfare Unfortunately, most of the genes involved
in disease resistance or susceptibility to disease are still largely unknown In addition to naturally occurring resistance genes, transgenes could be composed of genes that enhance immune response or in vitro-designed gene products (Table 2) Several approaches that have been investigated include transfer of genes for providing
Table 2 Naturally occurring disease resistance/susceptibility genes and in vitro designed genes conferring resistance
Encoding receptors for pathogens Antimicrobial peptides
Nonspecific immunity Enhancing the level and type of the immune response
(e.g., chemokines and cytokines) Encoding complement proteins Regulating phagocyte uptake and killing (e.g., NOS, Nramp) Specific (acquired) immunity Encoding receptors binding directly or indirectly to antigens
(T cell receptors, immunoglobulins, major histocompatibility complex, etc.) Mechanism Immunization (i.e., antibody production) DNA vaccines, immunoglobulin cDNAs
Interference with pathogen entry Recombinant pathogen receptors, coreceptors, etc
Interference with pathogen replication Antisense RNA, ribozymes, intrabodies (Source: Ref 10.)
Table 1 Some proposed modifications of milk constituents
Increase a and b
thermal stability, and increased calcium content Increase phosphorylation sites in caseins Increased calcium content,
improved emulsification Introduce proteolytic sites in caseins Increased rate of textural development to
improve cheese ripening
decreased micelle size, decreased gelation and coagulation Eliminate b
improved digestibility, decreased allergenic response, decreased primary source of cysteine in milk
decreased ice crystal formation, compromised osmotic regulation of mammary gland
and increased cheese yield
Decrease expression of acetyl CoA carboxylase Decreased fat content, improved nutritional quality,
reduced milk production costs Express immunoglobulin genes Protection against pathogens such as salmonella and listeria Replace bovine milk protein genes with human equivalents Mimic human breast milk
(Source: Refs 4 6.)
Trang 3resistance to influenza in pigs, preformed antibodies in
pigs, viral envelope proteins in chickens and pigs, and
antimicrobial peptides.[10]
As a first step toward enhancing mastitis resistance of
dairy animals, researchers generated transgenic mice that
secrete a potent antistaphylococcal protein, lysostaphin,
into milk.[11] Lysostaphin is a peptidoglycan hydrolase
normally produced by Staphylococcus simulans that is
active against Staphylococcus aureus bacteria S aureus
is the major contagious mastitis pathogen, accounting for
more than 15% of mastitis infections, and has proved
difficult to control using standard management practices
Three lines of transgenic mice were produced with an
ovine b
-lactoglobulin gene directing the secretion of
lysostaphin into milk Progeny of these mice exhibited
substantial resistance to an intramammary challenge of
S aureus, with the highest expressing line being
com-pletely resistant to infection These results clearly
demon-strated the potential of a transgene to combat one of the
most prevalent diseases of dairy cattle The same
lyso-staphin transgene has now been used to produce
trans-genic dairy cattle that are currently being evaluated
REDUCED ENVIRONMENTAL POLLUTION
In an effort to reduce phosphorus excretion in swine
manure, researchers at the University of Guelph[12]
constructed a transgene to provide expression of phytase
in salivary glands of pigs The saliva of these pigs contains
the phytase enzyme that allows the pigs to digest the
phosphorus in phytate, which is the most abundant source
of phosphorus in the pig diet Without this enzyme,
phosphorus in phytate passes undigested into feces to
become the single most important pollutant of swine
manure Their research showed that salivary phytase
essentially provides complete digestion of dietary phytate
phosphorus, relieves the requirement for inorganic
phosphate supplements, and reduces fecal phosphorus
output by up to 75% These pigs offer a unique biological
approach to the management of phosphorus nutrition and
reduce one of the major environmental pollutants
generated on swine farms
CONCLUSION
In the past few years, transgenic research to alter carcass
composition, increase milk production in sows, enhance
disease resistance, and reduce excretion of phosphate in
pigs has shown substantial progress Modification of milk
composition traits in dairy cattle offers considerable
potential, but much of this research is dependent upon
improving the efficiency of nuclear transfer, which will distinctly reduce the cost of producing transgenic cattle
REFERENCES
1 Palmiter, R.D.; Brinster, R.L.; Hammer, R.E.; Trumbauer, M.E.; Rosenfeld, M.G.; Birnberg, N.C.; Evans, R.M Dramatic growth of mice that develop from eggs micro injected with metallothionein growth hormone fusion genes Nature 1982, 300, 611 615
2 Pursel, V.G.; Rexroad, C.E., Jr Status of research with transgenic farm animals J Anim Sci 1993, 71 (Suppl 3),
10 19
3 Pursel, V.G.; Mitchell, A.D.; Wall, R.J.; Solomon, M.B.; Coleman, M.E.; Schwartz, R.J Transgenic Research to Enhance Growth and Lean Carcass Composition in Swine
In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 77 86
4 Jimenez Flores, R.; Richardson, T Genetic engineering of the caseins to modify the behavior of milk during processing: A review J Dairy Sci 1985, 71, 2640 2654
5 Yom, H C.; Bremel, R.D Genetic engineering of milk composition: Modification of milk components in lactating transgenic animals Am J Clin Nutr 1993, 58 (Suppl),
299 306
6 Maga, E.A.; Murray, J.D Mammary gland expression of transgenes and the potential for altering the properties of milk Bio/Technology 1995, 13, 1452 1457
7 Denning, C.; Burl, S.; Ainslie, A.; Bracken, J.; Dinnyes, A.; Fletcher, J.; King, T.; Ritchie, M.; Ritchie, W.A.; Rollo, M.; de Sousa, P.; Travers, A.; Wilmut, I.; Clark, A.J Deletion of the alpha (1,3) galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep Nat Biotechnol 2001, 19, 559 562
8 Noble, M.S.; Rodriguez Zas, S.; Cook, J.B.; Bleck, G.T.; Hurley, W.L.; Wheeler, M.B Lactational performance of first parity transgenic gilts expressing bovine alpha lactalbumin in their milk J Anim Sci 2002, 80, 1090 1096
9 Bawden, C.S.; McLaughlan, C.J.; Walker, S.K.; Speck, P.A.; Powell, B.C.; Huson, M.J.; Jones, L.N.; Rogers, G.E Improvement of Wool Quality by Transgenesis In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 67 76
10 Mu¨ller, M Increasing Disease Resistance in Transgenic Domestic Animals In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 87 97
11 Kerr, D.E.; Plaut, K.; Bramley, A.J.; Williamson, C.M.; Lax, A.J.; Moore, K.; Wells, K.D.; Wall, R.J Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice Nat Biotech nol 2001, 19, 66 70
12 Golovan, S.P.; Meidinger, R.G.; Ajakaiye, A.; Cottrill, M.; Wiederkehr, M.Z.; Barney, D.J.; Plante, C.; Pollard, J.W.; Fan, M.Z.; Hayes, M.A.; Laursen, J.; Hjorth, J.P.; Hacker, R.R.; Phillips, J.P.; Forsberg, C.W Pigs expressing salivary phytase produce low phosphorus manure Nat Biotechnol 2001, 19, 741 745
Trang 4Transgenic Animals: Modifying the
Mitochondrial Genome
Carl A Pinkert
University of Rochester Medical Center, Rochester, New York, U.S.A
Lawrence C Smith
Universite´ de Montre´al, Quebec, Canada
Ian A Trounce
University of Melbourne, Victoria, Australia
INTRODUCTION
In comparison to the techniques successfully employed
for nuclear gene transgenesis in livestock over the past 20
years, the lack of comparable recombination in
mitochon-drial DNA (mtDNA) has, until recently, prevented its
direct in vivo manipulation The coordinated expression
of single-copy nuclear gene products, together with the
polyploid mtDNA gene products, is required for normal
mitochondrial biogenesis and respiratory chain function
It is of great current interest to seek improved
technol-ogies for manipulating the mitochondrial genome, so that
interactions of nuclear and mtDNA genotypes can be
studied in experimental systems
MITOCHONDRIAL GENETICS
AND ANIMAL MODELING
Mammalian mitochondria contain between one and
approximately ten copies of a closed, circular,
super-coiled, double-stranded DNA that is bound to the inner
mitochondrial membrane and is not associated with
histones or a scaffolding protein matrix The mtDNAs of
all vertebrates are highly conserved and quite small
( 16.5 kb in length) in comparison to the nuclear
genome Mammalian mitochondria have their own genetic
systems, replete with a unique genetic code, genome
structure, transcriptional and translational apparatus, and
tRNAs Perhaps, because of a postulated less-extensive
mitochondrial DNA (mtDNA) repair system and because
of the absence of protective histones, the mitochondrial
genome is subject to an increased sensitivity to mutations
due to metabolic (e.g., oxidative stress) and environmental
(e.g., toxins, mutagens, and UV light) sources
Mitochon-drial genes encode for 13 of the protein subunits that
function in the mitochondrial oxidative phosphorylation
system, along with two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs) Accordingly, directed modifica-tion of mitochondrial genes and/or their funcmodifica-tion would provide a powerful tool in production agriculture.[1] Cytoplasmic-based traits in domestic animals have included growth, reproduction, and lactation In addition, mitochondrial restriction fragment-length polymorphisms (RFLPs) were identified and associated with specific lactational characteristics in a number of dairy cattle lineages The matrilineal inheritance of mammalian mtDNA has also been used to advantage in studies exploring the timing and geography of domestication events, as recently demonstrated for horses, where multiple domestication events appear to have occurred in the Eurasian steppe.[2] In addition, metabolic and cellular abnormalities in humans were correlated to mutations arising exclusively within the mitochondrial genome Indeed, various diseases have been associated with mtDNA point mutations, deletions, and duplications (e.g., diabetes mellitus, myocardiopathy, and retinitis pigmentosa) as well
as age-associated changes in the functional integrity of mitochondria (as seen in Parkinson’s, Alzheimer’s, and Huntington’s diseases) As such, for both agricultural and biomedical research efforts, the ability to manipulate the mitochondrial genome and to regulate the expression of mitochondrial genes would provide one possible mode of genetic manipulation and therapy
The creation of heteroplasmic transmitochondrial animals has developed along three lines: 1) direct mito-chondrial injection into oocytes or embryos; 2) embryonic stem (ES) cell-based technologies; and 3) in relation to karyoplast or cytoplast transfer (including consequences associated with nuclear transfer or cloning experimenta-tion; Table 1) These techniques have illustrated model systems that will provide a greater understanding of mito-chondrial dynamics, leading to the development of ge-netically engineered production animals, and therapeutic
DOI: 10.1081/E EAS 120024365
Trang 5strategies for human metabolic diseases affected by
aberrations in mitochondrial function
As described in a number of recent reports,[3,4]
nuclear-encoded genes and knock-out modeling have been
informative in identifying novel models in mitochondrial
disease pathogenesis as well as critical pathways
associ-ated with mitochondrial function With initial
character-ization of these nuclear gene-encoded models, our search
for a greater understanding of mitochondrial interactions
and function would eventually lead us to a desire to
develop methodology for mitochondria and mitochondrial
gene transfer As a first step, efficient methods to
introduce foreign or altered mtDNA or genomes into
somatic or germ cells would be needed
TRANSMITOCHONDRIAL ANIMALS
To make a transmitochondrial animal, the ability to
manipulate normal and mutant mitochondria in vivo has
been a critical and difficult first step In vivo
mitochon-drial gene transfer remains a technological hurdle in the
development of mitochondria-based genetic therapies and
in the generation of experimental animal models for the
study of mitochondrial dynamics and mitochondria-based
traits While gene transfer has been performed in a host of
cell types and organisms, transfer of nuclear DNA has
been the only demonstrable form of mammalian gene
transfer, short of cell fusions, to date
Rapid segregation of mtDNA genotypes could occur in
mammals and was first demonstrated in Holstein cattle
where pedigree records in the industry allowed detailed
analysis of maternally related individual genotypes.[5]
Segregation of mtDNA was investigated in maternal
lineages of heteroplasmic mice created by cytoplast
fusion[6,7]and by embryonic karyoplast transplantation.[8]
Although mitochondrial segregation in somatic tissues is
effective in some tissues and with increasing age, the preceding studies have shown that mtDNA heteroplasmy
is maintained at stable levels throughout several gen-erations This would suggest that the mouse germline
is not very effective in segregating mtDNA haplotypes
In cattle, however, highly heteroplasmic females will produce homoplasmic oocytes, whereas heteroplasmic bulls produce mostly heteroplasmic sperm, indicating that mtDNA segregation is very stringent in the female and practically absent in the male germline.[9]Together, these results suggest that mammalian species show variable patterns of mtDNA segregation
In contrast to these techniques, our efforts to devise a direct mitochondria transfer technique offered certain advantages Principally, the ability to use isolated mito-chondria for the production of heteroplasmic mice would allow for investigations into the feasibility of genetic manipulation of mtDNA in vitro prior to mitochondria microinjection into zygotes
CONCLUSIONS
Through the early 1990s, various early attempts to create transmitochondrial strains of mammalian species by introduction of foreign mitochondria into germ cells were largely unsuccessful A number of constraints have been identified or postulated, from perturbations of biological pathways to mechanistic aspects of the specific protocols used Since 1997, a number of laboratories have reported
on methodologies used to create transmitochondrial animals To date, methods for mitochondria isolation and interspecific transfer of mitochondria have been reported both in laboratory and domestic animal models.[3,10,11] Interestingly, early reports on development of cloned animals by nuclear transfer resulted in conflicting con-sequences when retrospective studies on mitochondrial
Table 1 Methods for creating mitochondrial modifications in animals
Method
Heteroplasmy/
homoplasmy detected
Germline
Karyoplast fusion
(nuclear transfer)
PEG or electrofusion Karyoplast or cytoplast
transfer into ES cells and transfer
competent/efficient cell lines
low level heteroplasmy
or programmed destruction postfertilization
Trang 6transmission were reported.[12–16]Indeed, dependent upon
the specific methodology employed for nuclear transfer
and cytoplasm/ooplasm transfer to rescue low-quality
embryos, additional models of heteroplasmy may or may
not have been characterized as a consequence of
mitochondrial dysfunction As such, research independent
of targeted mitochondrial genomic modifications may also
help unlock mechanisms underlying the dynamics related
to persistence of foreign mitochondria and maintenance of
heteroplasmy in various cloning protocols
ARTICLES OF FURTHER INTEREST
Biotechnology: Stem Cell and Germ Cell Technology,
p 146
Biotechnology: Transgenic Animals, p 149
Contributions to Society: Biomedical Research Models,
p 239
Genetics: Molecular, p 466
Molecular Biology: Animal, p 653
Transgenic Animals: Improved Performance, p 837
REFERENCES
1 Pinkert, C.A Genetic Engineering of Animals In Hand
book of Biomedical Technology and Devices; Moore, J.E.,
Jr., Zouridakis, G., Eds.; CRC Press: Boca Raton, 2004;
18 1 18 12
2 Vila, C.; Leonard, J.A.; Gotherstrom, A.; Marklund, S.;
Sandberg, K.; Liden, K.; Wayne, R.K.; Ellegren, H
Widespread origins of domestic horse lineages Science
2001, 291 (5503), 474 477
3 Pinkert, C.A.; Trounce, I.A Production of transmitochon
drial mice Methods 2002, 26 (4), 348 357
4 Wallace, D.C Mouse models for mitochondrial disease
Am J Med Genet 2001, 106 (1), 71 93
5 Olivo, P.D.; Van de Walle, M.J.; Laipis, P.J.; Hauswirth,
W.W Nucleotide sequence evidence for rapid genotypic
shifts in the bovine mitochondrial DNA D loop Nature
1983, 306 (5941), 400 402
6 Jenuth, J.P.; Peterson, A.C.; Fu, K.; Shoubridge, E.A
Random genetic drift in the female germline explains the
rapid segregation of mammalian mitochondrial DNA Nat Genet 1996, 14 (2), 146 151
7 Jenuth, J.P.; Peterson, A.C.; Shoubridge, E.A Tissue specific selection for different mtDNA genotypes in heteroplasmic mice Nat Genet 1997, 16 (1), 93 95
8 Meirelles, F.V.; Smith, L.C Mitochondrial genotype segregation in a mouse heteroplasmic lineage produced
by embryonic karyoplast transplantation Genetics 1997,
145(2), 445 451
9 Smith, L.C.; Bordignon, V.; Garcia, J.M.; Meirelles, F.V Mitochondrial genotype segregation and effects during mammalian development: Applications to biotechnology Theriogenology 2000, 53 (1), 35 46
10 Meirelles, F.V.; Bordignon, V.; Watanabe, Y.; Watanabe, M.; Dayan, A.; Lobo, R.B.; Garcia, J.M.; Smith, L.C Compete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte Genetics 2001, 158 (1), 351 356
11 McKenzie, M.; Trounce, I.A.; Cassar, C.A.; Pinkert, C.A Production of homoplasmic xenomitochondrial mice Proc Natl Acad Sci USA 2004, 101 (6), 1685 1690
12 Hiendleder, S.; Zakhartchenko, V.; Wenigerkind, H.; Reichenbach, H.D.; Bruggerhoff, K.; Prelle, K.; Brem, G.; Stojkovic, M.; Wolf, E Heteroplasmy in bovine fetuses produced by intra and inter subspecific somatic cell nuclear transfer: Neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood Biol Reprod
2003, 68 (1), 159 166
13 Evans, M.J.; Gurer, C.; Loike, J.D.; Wilmut, I.; Schnieke, A.E.; Schon, E.A Mitochondrial DNA genotypes in nuclear transfer derived cloned sheep Nat Genet 1999,
23(1), 90 93
14 Hiendleder, S.; Schmutz, S.M.; Erhardt, G.; Green, R.D.; Plante, Y Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle Mol Reprod Dev 1999, 54 (1), 24 31
15 Steinborn, R.; Schinogl, P.; Zakhartchenko, V.; Achmann, R.; Schernthaner, W.; Stojkovic, M.; Wolf, E.; Muller, M.; Brem, G Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning Nat Genet
2000, 25 (3), 255 257
16 Takeda, K.; Takahashi, S.; Onishi, A.; Goto, Y.; Miya zawa, A.; Imai, H Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer J Reprod Fertil 1999,
116(2), 253 259
Trang 7Transgenic Animals: Secreted Products
Michael J Martin
David A Dunn
Carl A Pinkert
University of Rochester Medical Center, Rochester, New York, U.S.A
INTRODUCTION
Interest in modifying traits that determine productivity of
domestic animals was greatly stimulated by early
experi-ments in which body size and growth rates were
dramatically affected in transgenic mice expressing
growth hormone transgenes driven by a metallothionein
(MT) enhancer/promoter From that starting point, similar
attempts followed to enhance growth in farm animals by
introduction of various growth factors, modulators, and
their receptors It soon became apparent that transgene
regulation was an exquisite balancing act, where precise
regulation of transgenes was crucial to normal
devel-opment Yet, the overexpression of various transgene
products illustrated that such animals could produce
biologically important molecules as efficient mammalian
bioreactors, with efficiencies far greater than conventional
bacterial or cell culture systems From early studies in the
mid-1980s through the 1990s, one of the main targets of
genetic engineering or gene pharming efforts has involved
attempts to direct expression of transgenes encoding
biologically active human proteins in farm animals To
date, expression of foreign genes encoding various protein
products was successfully targeted to the mammary
glands of goats, sheep, cattle, and swine, yet the jump
from model to achieving regulatory approval has proven
most challenging
ADVANTAGES OF RECOMBINANT PROTEIN
SYNTHESIS IN TRANSGENIC ANIMALS
Several different organisms have been harnessed to
produce recombinant proteins Bacteria, yeast, fungi,
plants, and cultured mammalian cells can all be
reprogrammed and, if properly managed, yield relatively
large amounts of recombinant proteins Problems begin to
arise, however, when one examines the ability of these
organisms to posttranslationally modify and even release
recombinant proteins Bacteria, for example, are often
unable to package and secrete recombinant proteins In
these instances, the recombinant protein must be
physi-cally extracted from the bacteria, a process that can be difficult and costly Whereas yeast can secrete recombinant proteins that are glycosylated, the enzymatic pathway(s) that they utilize to accomplish protein glycosylation differs from that employed in higher plants and animals
As a result, many of the recombinant proteins produced
by yeast exhibit inadequate glycosylation Posttrans-lational modification of recombinant proteins produced
in fungi appears to be aberrant in many instances as well Mammalian cell lines, in contrast, typically perform posttranslational modifications of recombinant proteins that are quite similar to those observed in indigenous proteins Primary drawbacks to the synthesis of recom-binant proteins in animal cell lines include cost and the logistical challenge associated with developing and managing cell cultures for large-scale protein production
In contrast, transgenic animals, as Louis-Marie Hou-debine describes,[1] share most of the properties of animal cells in culture, exhibit appropriate posttransla-tional modifications of recombinant proteins, and synthe-size and secrete proteins extremely efficiently Indeed, mammary gland epithelia typically have a cell density that is 100- to 1000-fold greater than that used in mam-malian cell culture bioreactors In one recent example, 35 transgenic goats that produced a human monoclonal antibody at a concentration of 8 g/L in their milk were equivalent to an 8500-liter batch cell culture running
200 days/year with a 1 g/l final production level.[2]Thus, from a production standpoint, the amount of antibody synthesized in 170,000-liter cell culture yield was equiv-alent to that generated in 21,000 liters of milk from transgenic goats Assuming a process yield of 60%, both systems would generate 100 kg of purified monoclonal antibody, yet the transgenic bioreactor was significantly more efficient
Another obvious incentive for the production of biopharmaceuticals in transgenic livestock is their poten-tial economic value (Table 1) The cost of human proteins obtained from donated plasma and used in replacement therapy has ranged from $4/g for serum albumin and
$5000/g for antithrombin III to $150,000/g for human blood clotting factor VIII (FVIII).[4] Although the individual values of these seem dramatic, they pale in
DOI: 10.1081/E EAS 120019825
Trang 8comparison to the projected worth of a number of
recombinant structural products Biomedical applications
of BiosteelTM(Nexia Inc.), a recombinant form of dragline
spider silk, produced in the milk of transgenic goats, is
projected to represent $150 to $450 million in annual
earnings (exclusive of military and other
industri-al applications)
EXPRESSION OF RECOMBINANT
PROTEINS IN MILK
Since the introduction of the first exogenous genes into
mice, more than 60 proteins have been produced in milk
of transgenic animals In order to target protein expression
specifically to the mammary gland, a transgene typ-ically consists of the desired protein gene fused to one
of several available mammary-specific regulatory se-quences.[3–7]These sequences have included: ovine BLG; murine, rat, and rabbit whey acidic protein (WAP); bovine
a-s1casein; rat, rabbit, and goatb-casein; and guinea pig, ovine and caprine, and bovine a-lactalbumin While ex-pression of the target protein can be achieved using either a genomic DNA or cDNA coding sequence(s), the former normally yields higher levels of protein expression Therapeutic monoclonal antibodies produced in the mammary gland of a transgenic animal line present a potentially valuable technology Transgenic monoclonal antibodies are produced by cloning genetic sequences for both heavy- and light-chain genes downstream of
Table 1 Molecular pharming projects: Potential biomedical and commercial products from transgenic farm animals
a fetoprotein (rhAFP) Myasthenia gravis, multiple sclerosis,
and rheumatoid arthritis
(Merrimack/GTC)
Phytase (EnviropigTM
Spider silk (Biosteel1
Monoclonal antibodies and immunoglobulin fusion proteins:
AntegrenTM
and rheumatoid arthritis
(Merrimack /GTC)
(Adapted from Ref 3.)
Trang 9mammary gland-specific regulatory elements Chimeric
antibodies may also be produced by ligating
antigen-binding region sequences from a (usually murine)
monoclonal antibody to constant region sequences from
a different species and/or isotype The first transgenic
mice harboring immunoglobulin genes were made in the
mid-1980s.[8]Though the majority of effort and funding in
this field is currently focused toward human therapeutics,
veterinary use of monoclonal antibodies also shows
significant promise as a developing application
Whereas several therapeutic monoclonal antibodies
have been approved for use by the U.S Food and Drug
Administration, none as yet has been approved where a
transgenic animal was used as a production vehicle Using
antibody production technologies in transgenic
biore-actor systems, these products target a wide range of
clinical ailments and are mostly in the preclinical stage
of development
EXPRESSION OF RECOMBINANT PROTEINS
IN MEDIA OTHER THAN MILK
Secretion of transgene-encoded proteins in the urine of
transgenic animals was demonstrated using recombinant
genes under the control of kidney-[9] or bladder-[10]
specific regulatory sequences Expression of transgenes in
the kidney or bladder of transgenic animals and
subsequent secretion in the urine may provide some
advantages over the mammary gland as a bioreactor, as
the purification of proteins from urine may be facilitated
by lower lipid and protein levels in comparison to milk
Additionally, such animals can be used for production
of recombinant proteins over the course of their entire
life span
RECOMBINANT PROTEIN PRODUCTION:
HEALTH AND SAFETY ISSUES
In addition to being structurally and functionally
analo-gous to the natural plasma-derived protein, purified
recombinant proteins must be free of pathogenic
organ-isms Viral and bacterial contamination of human
biopharmaceutical products produced in the blood or
milk can be minimized by focusing prevention/eradication
efforts on at least three levels of production: the transgenic
animal donor, the medium in which the recombinant
protein is produced, and the final product.[4]An initial key
to minimizing the risk of contamination is to derive the
transgenic donor animals from a source herd that is free
from as many pathogens as possible Maintenance of these
animals in a closed facility, the implementation of strict
monitoring procedures for various pathogens, and the use
of animal husbandry practices that follow generally accepted practices (GAPs) and standard operating proce-dures should greatly reduce the entry of pathogens Though quite costly, one can develop pathogen-free herds
of transgenic livestock Such a feat was recently achieved
by introducing hysterotomy-derived transgenic piglets into an elaborate SPF barrier facility.[11] Diagnostic testing of this herd over the past 3 years in this facility had revealed the absence of 35 major and minor swine pathogens including PRRS, parvovirus, leptospira, para-influenza, and Streptococcus suis
CONCLUSION
While transgenic animal technology continues to open new and unexplored agricultural frontiers, molecular pharming efforts raise questions concerning regulatory and commercialization issues Although significant advances have been made since the inception of various clinical trials, the resources required to move the projects forward and the attendant financial risks have led a number of companies to curtail product development Various societal issues exist and will continue to influence the development of value-added animal products pro-duced through transgenesis until transgenic products and foodstuffs are proven safe for human use and are accepted by a wide cross section of society
ARTICLES OF FURTHER INTEREST
Biotechnology: Stem Cell and Germ Cell Technology,
p 146 Biotechnology: Transgenic Animals, p 149 Contributions to Society: Biomedical Research Models,
p 239 Genetics: Molecular, p 466 Molecular Biology: Animal, p 653 Overall Contributions of Domestic Animals to Society,
p 696 Phytases, p 704 Proteins, p 757 Transgenic Animals: Improved Performance, p 837
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