Review TRENDS in Biotechnology Vol.22 No.6 June 2004
The contribution of farm animals to
human health
Wilfried A. Kues and Heiner Niemann
Department of Biotechnology, Institut für Tierzucht, Mariensee, D-31535 Neustadt, Germany
Farm animals and their products have a longstanding history. The pioneering work of Edward Jenner with
and successful history of providing significant contri- cowpox in the 18th century paved the way for modern
butions to human nutrition, clothing, facilitation of vaccination programs against smallpox, as well as other
labour, research, development and medicine and have human and animal plagues. To date, more than 250 million
thus been essential in improving life expectancy and people have benefited from drugs and vaccines produced
human health. With the advent of transgenic technol- by recombinant technologies in bacteria and various types
ogies the potential of farm animals for improving of mammalian cells and many more will benefit in the
human health is growing and many areas remain to be future (New Medicines in Development for Biotechnology,
explored. Recent breakthroughs in reproductive tech- 2002; www.phrma.org/newmedicines/biotech/). Further
nologies, such as somatic cloning and in vitro embryo examples of the significant contribution of farm animals
production, and their merger with molecular genetic to human health are the longstanding use of bovine and
tools, will further advance progress in this field. Here, porcine insulin for treatment of diabetes as well as horse
we have summarized the contribution of farm animals antisera against snake venoms and antimicrobial pep-
to human health, covering the production of antimicro- tides. In addition, farm animals are models for novel
bial peptides, dietary supplements or functional foods, surgical strategies, testing of biodegradable implants and
animals used as disease models and the contribution of sources of tissue replacements, such as skin and heart
animals to solving urgent environmental problems and valves.
challenges in medicine such as the shortage of human Progress in transgenic technologies has allowed the
cells, tissues and organs and therapeutic proteins. generation of genetically modified large animals for
Some of these areas have already reached the level of applications in agriculture and biomedicine, such as the
preclinical testing or commercial application, others production of recombinant proteins in the mammary gland
will be further advanced only when the genomes of the and the generation of transgenic pigs with expression of
animals concerned have been sequenced and anno- human complement regulators in xenotransplantation
tated. Provided the necessary precautions are being research [1]. Further promising application perspectives
taken, the transmission of pathogens from animals to will be developed when somatic cloning with genetically
humans can be avoided to provide adequate security. modified donor cells is further improved and the genomes
Overall, the promising perspectives of farm animals and of farm animals are sequenced and annotated. The first
their products warrant further research and develop- transgenic livestock were born less than 20 years ago with
ment in this field. the aid of microinjection technology [2]. Recently the first
animals with knockout of one or even two alleles of a
Farm animals have made significant contributions to targeted gene were reported (Table 1). Somatic nuclear
human health and well-being throughout mankind s transfer has been successful in 10 species, but the overall
Table 1. Milestones (live offspring) in transgenesis and reproductive technologies in farm animals
Year Milestone Strategy Refs
1985 First transgenic sheep and pigs Microinjection of DNA into one pronucleus of a zygote [2]
1986 Embryonic cloning of sheep Nuclear transfer using embryonic cells as donor cells [91]
1997 Cloning of sheep with somatic donor cells Nuclear transfer using adult somatic donor cells [92]
1997 Transgenic sheep produced by nuclear transfer Random integration of the construct [93]
1998 Transgenic cattle produced from fetal fibroblasts and nuclear transfer Random integration of the construct [94]
1998 Generation of transgenic cattle by MMLV injection Injection of oocytes with helper viruses [95]
2000 Gene targeting in sheep Gene replacement and nuclear transfer [96]
2002 Trans-chromosomal cattle Additional artificial chromosome [15]
2002 Heterozygous knockout in pigs One allele of a-galactosyl-transferase knocked out [34,35]
2003 Homozygous gene knockout in pigs Both alleles of a-galactosyl-transferase knocked out [36]
2003 Transgenic pigs via lentiviral injection Gene transfer into zygotes via lentiviruses [97]
Corresponding author: Heiner Niemann ([e-mail]).
www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.04.003
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Table 2. Efficiency of somatic cloning of mammals
Species Number of animalsa % Viable offspring Comments
Cattle ,3000 15 20 Up to 30% of the cloned calves showed abnormalities, such as increased birth weight
Sheep ,400 5 8 Same problems as with cattle clones
Goat ,400 3 Minor health problems reported
Mouse ,300 ,2 Some adult mice clones showed obesity and a reduced life span
Pig ,200 ,1 Some cloned piglets had reduced birth weights
Cat 1 ,1
Rabbit 6 ,1
Mule 1 ,1
Horse 1 ,1
Rat 2 ,1
a
Estimated total numbers of mammals derived from somatic cloning since 1997.
efficiency is low and few cloned offspring have been born correct glycosylation patterns and post-translational
worldwide (Table 2). Compared with microinjection of modifications, low running costs, rapid propagation of
DNA constructs into pronuclei of zygotes, somatic nuclear the transgenic founders and high expression stability.
transfer is superior for the generation of transgenic These attractive perspectives led to the development of the
animals (Table 3).  gene pharming concept, which has been advanced to the
Here, we have summarized the contribution of farm level of commercial application [5]. The most promising
animals to human health covering (i) the production of site for production of recombinant proteins is the mam-
pharmaceuticals; (ii) production of xenografts for over- mary gland, but other body fluids including blood, urine
coming the severe shortage of human organs and tissues; and seminal fluid have also been explored [6]. The
(iii) the use of farm animals as disease models; (iv) the mammary gland is the preferred production site mainly
production of dietary supplements or functional foods; and because of the quantities of protein that can be produced
(v) the contribution of farm animals to solving environ- and the ease of extraction or purification of the respective
mental problems. protein.
Based on the assumption of average expression levels,
Farm animals for pharmaceutical production daily milk volumes and purification efficiency, 5400 cows
Gene  pharming : production of recombinant human would be needed to produce the 100 000 kg of human
proteins in the mammary gland of transgenic animals serum albumin (HSA) that are required per year world-
The conventional production of rare human therapeutic wide, 4500 sheep would be required for the production of
proteins from blood or tissue extracts is an inefficient, 5000 kg a-antitrypsin (a-AT), 100 goats for 100 kg of
expensive, labour and time consuming process, which in monoclonal antibodies, 75 goats for the 75 kg of antith-
addition bears the risk of contamination with human rombin III (ATIII) and two pigs to produce 2 kg human
pathogens. The production of human therapeutic proteins clotting factor IX. All these values are calculated on a
by recombinant bacteria or cell cultures has alleviated yearly basis [3].
these problems and has made several therapeutic proteins Large amounts of numerous heterologous recombinant
available for patients. However, these recombinant sys- proteins have been produced by targeting expression to the
tems have several limitations. They are only suitable for mammary gland via mammary gland-specific promoter
 simple proteins, the amount of protein produced is elements. Proteins were purified from the milk of
limited, and post-translational modifications are often transgenic rabbits, pigs, sheep, goats and cattle. The
incorrect leading to immune reactions against the protein. biological activity of the recombinant proteins was
In addition, the technical prerequisites are challenging assessed and therapeutic effects have been characterized
and production costs are high. [3,7]. Products such as ATIII, a-AT or tissue plasminogen
Farm animals such as cattle, sheep, goats, pigs and activator (tPA) are advanced to clinical trials (Table 4) [5].
even rabbits [3,4] have several significant advantages for Phase III trials for ATIII have been completed and the
the production of recombinant proteins over other sys- protein is expected to be on the market within the next 2 3
tems, including their potential for large-scale production, years. In February 2004 an application was submitted to
Table 3. Advantages and disadvantages of two gene transfer methodologiesa
Microinjection Somatic nuclear transfer
Integration efficiency þþþþ
Integration site Random Random or targeted
Gene deletion 2 þþþ
Construction size .50 kb (Artificial chromosomes) , 30 50 kb
Technical feasibility Technically demanding Technically demanding
Mosaicism þþþ 2
Expression screen in vitro þþþþ
Expression pattern Variable Controlled, consistent
Multi-transgenics þþþþ
a
Abbreviations: 2, not possible; þ, weak advantage; þþ, moderate advantage; þþþ, strong advantage
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Table 4. Proteins produced in the mammary gland of transgenic farm animalsa
Protein Developmental phase Production species Therapeutic application Potential market introduction date
AT III Phase III Goat Genetic heparin resistance 2005
TPA Phase II/III Goat Dissolving coronary clots . 2006
a-AT Phase II/III Goat and/or sheep Lung emphysema . 2007
Cystic fibrosis
hFVIII Experimental Sheep Hemophilia A . 2008
HAS Phase I Cattle Blood substitute . 2008
Various antibodies Phase I/II Goat . 2007
a
Abbreviations: a-AT, a-A1-antitrypsin; AT III, antithrombin III; hFVIII, human clotting factor VIII; HSA, human serum albumin; TPA, tissue plasminogen activator
the European Market Authorization to allow Atrynw, the chromosome (HAC) containing the entire sequences of
recombinant ATIII from the milk of transgenic dairy goats, the human immunoglobulin heavy and light chain loci
to enter the market as a fully registered drug. The enzyme has been introduced into bovine fibroblasts, which
a-glucosidase from the milk of transgenic rabbits has were then used in nuclear transfer. Transchromosomal
Orphan drug registration and has been successfully used offspring were obtained that expressed human
for the treatment of Pompe s disease [8]. This is a rare immunoglobulin in their blood. This system could be
glycogen storage disorder, which is fatal in children under a significant step forward in the production of human
2 years and currently application with recombinant therapeutic polyclonal antibodies [15]. Further studies
a-glucosidase is the only way to treat this metabolic defect. will show whether the additional chromosome will be
Biologically active human lactoferrin has been produced in maintained over future generations and how stable
large amounts in the mammary glands of transgenic cows expression will be.
and will probably be developed as a biopharmaceutical for
prophylaxis and treatment of infectious diseases [9]. Production of a new class of antibiotics: cationic anti-
Guidelines developed by the Food and Drug Adminis- microbial peptides
tration (FDA) of the USA require monitoring of the With increasing antibiotic resistance in bacterial species,
animals health, validation of the gene construct, charac- there is a growing need to develop new classes of anti-
terization of the isolated recombinant protein, as well as microbial agents. Cationic anti-microbial peptides (AMP)
have many of the desired features [16] because they
performance of the transgenic animals over several
generations. This has been taken into account when possess a broad spectrum of activity, kill gram-positive and
gram-negative bacteria rapidly, are unaffected by classical
developing  gene pharming , for example by using only
animals from prion disease-free countries (New Zealand) resistance genes and are active in animal models [17 19].
and keeping the animals in very hygienic conditions. AMPs belong to the innate immune defense, which acts as
Successful drug registration of Atrynw will demonstrate a first barrier ahead of humoral and cellular immune
the usefulness and solidity of this approach and will systems, and neutralizes bacteria by interacting specifi-
accelerate registration of further products from this cally with their cell membranes. Their low transmem-
process, as well as stimulate research and commercial brane potential of ,-100 mV, and the abundant anionic
activity in this area. phospholipids are essential for this selective interaction. It
When considering the  gene pharming concept, one has is proposed that AMPs physically disintegrate the cell
to bear in mind that not every protein can be expressed at membrane, and in addition can interact with several
the desired levels. Erythropoietin (EPO) could not be intracellular target molecules [16]. More than 500 such
expressed in the mammary gland of transgenic cattle [10] peptides have been discovered in plants, insects, inverte-
and was even detrimental to the health of rabbits brates, fish, amphibians, birds and mammals [16 20].
transgenic for EPO [11]. We have shown that human AMPs from livestock species would be superior anti-
clotting factor VIII (hFVIII) cDNA constructs can be microbial drugs because they would lack cytotoxic effects
expressed in the mammary gland of transgenic mice, that were found for insect peptides; the evolution of
rabbits and sheep [1,12]. However, the yields of biologically resistance would not affect the human specific innate
active recombinant hFVIII protein from ovine milk were immunity [20].
low because hFVIII was rapidly sequestered into ovine Prominent examples of cationic anti-microbial peptides
milk. [13]. These results show that the technology needs from farm animals already in advanced clinical trials
further improvements to achieve high-level expression (Phases II III) are Iseganan (derived from protegrin-1
with large genes having complex regulation, such as that peptide of pig leucocytes; Intrabiotics; http://intrabiotics.
coding for hFVIII, although higher levels of hFVIII have com/) and MBI-594 (similar to indolicidin peptide from
been reported in transgenic swine [14]. With the advent of bovine neutrophils; Micrologix, http://mbiotech.com/). To
transgenic crops that produce pharmacologically active date, there are no documented cases of antimicrobial
proteins, there is an array of recombinant technologies peptide-resistance for AMPs and combinatorial
available that will allow the most appropriate production approaches in peptides (20 possible amino acids in each
system for a specific protein to be targeted. position) provide great potential for rational drug design
An interesting new development is the generation of [21]. Recombinant production will keep the production
transchromosomal animals (Table 1). A human artificial costs low.
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Xenotransplantation of porcine organs to human occurs within seconds or minutes. In the case of a
patients discordant organ (e.g. in transplanting from pig to
Solid organs human) naturally occurring antibodies react with anti-
Today .250 000 people are alive only because of the genic structures on the surface of the porcine organ and
successful transplantation of an appropriate human organ induce HAR by activating the complement cascade via the
antigen antibody complex. Ultimately, this results in the
(allotransplantation). On average, 75 90% of patients
formation of the membrane attack complex (MAC).
survive the first year after transplantation and the
However, the complement cascade can be shut down at
average survival of a patient with a transplanted heart,
various points by expression of regulatory genes that
liver or kidney is 10 15 years. This progress in organ
prevent the formation of the MAC. Regulators of the
transplantation technology has led to an acute shortage of
complement cascade are CD55 (decay accelerating factor,
appropriate organs, and cadaveric or live organ donation
DAF), CD46 (membrane cofactor protein, MCP) or CD59.
cannot cover the demand in western societies. The 2001
MAC disrupts the endothelial cell layer of the blood
figures from the United Network for Organ Sharing (www.
vessels, which leads to lysis, thrombosis, loss of vascular
unos.org) in the USA show that the ratio of patients with
integrity and ultimately to rejection of the transplanted
organ transplantation to those on the waiting list is ,1:4
organ [24].
(Table 5). A similar ratio is found in other countries such as
Induced xenoreactive antibodies are thought to be
the UK, France and Germany. A new person is added to the
responsible for AVR, which occurs within days of a
waiting list every 14 minutes. This has led to the sad and
transplantation of a xenograft; disseminated intravascu-
ethically challenging situation in which several thousand
lar coagulation (DIC) is a predominant feature of AVR.
patients who could have survived if appropriate organs
Despite severe immunosuppressive treatment, a disturbed
had been available die every year.
thrombocyte function and DIC were observed in a pig-to-
To close the growing gap between demand and
primate xenotransplant model [25,26]. The endothelial
availability of appropriate organs, porcine xenografts are
cells of the graft s microvasculature loose their anti-
considered the solution of choice [22,23]. Today the
thrombic properties, attract leucocytes, monocytes and
domesticated pig is considered the optimal donor animal
platelets leading to anemia and organ failure. The
because (i) the organs are similar in size to human organs;
underlying mechanism for DIC and thrombotic micro-
(ii) porcine anatomy and physiology are not too different
angiopathy is thought to be activation of the endothelial
from that of humans; (iii) pigs have short reproduc-
cells attributed to incompatibilities between human and
tion cycles and large litters; (iv) pigs grow rapidly;
porcine coagulation factors [25]. At least three incompat-
(v) maintenance of high hygienic standards is possible at
ibilities between human and porcine coagulation systems
relatively low costs; and (vi) transgenic techniques for
have been identified; the first is the failure of porcine
modifying the immunogenicity of porcine cells and organs
thrombomodulin (TM) to activate human anticoagulant
are well established.
protein C, the second is that the porcine tissue factor
The process of generating and evaluating transgenic
pathway inhibitor fails to inhibit human clotting factor Xa
pigs as potential donors for xenotransplants involves a
and the third is that porcine von Willebrand factor (vWF)
variety of complex steps and is time-, labour- and resource-
binds and activates human platelets [26]. Human throm-
intensive.
bomodulin (hTM) and heme-oxygenase 1 (hHO-1) are
Essential prerequisites for successful xenotransplanta-
crucially involved in the etiology of DIC and might be good
tion are:
targets for future transgenic studies to improve long-term
(i) Overcoming the immunological hurdles.
survival of porcine xenografts by creating multi-trans-
(ii) Prevention of transmission of pathogens from the
genic pigs.
donor animal to the human recipient.
The cellular rejection occurs within weeks after
(iii) Compatibility of the donor organs with the human
transplantation. In this process the blood vessels of the
organ in terms of anatomy and physiology.
transplanted organ are damaged by T-cells, which invade
The immunological obstacles in a porcine-to-human
the intercellular spaces and destroy the organ. This
xenotransplantation are the hyperacute rejection
rejection is observed after allotransplantation and is
response (HAR), acute vascular rejection (AVR), cellular
normally suppressed by life-long administration of immu-
rejection and potentially chronic rejection [24]. The HAR
nosuppressive drugs [24].
When using a discordant donor species such as the pig,
Table 5. Overview of transplanted organs and demand for
overcoming the HAR and AVR are the preeminent goals.
organ transplantation (USA)a
The most promising strategy for overcoming the HAR is
the synthesis of human complement regulatory proteins
Type of transplant Transplantations in 2001 Waiting patients
(RCAs) in transgenic pigs [22,23,27,28]. Following trans-
Kidney 14 152 52 772
Liver 5177 17 520
plantation, the porcine organ would produce the comp-
Pancreas 468 1318
lement regulatory protein and can thus prevent the
Kidney/Pancreas 884 2520
complement attack of the recipient. Pigs transgenic for
Heart 2202 4163
DAF or MCP have been generated by microinjection of
Heart/Lung 27 209
Lung 1054 3799 DNA constructs into pronuclei of zygotes. Hearts and
Total 23 964 79 845
kidneys from these animals have been transplanted either
a
Data from United Network for Organ Sharing, 2001 (www.unos.org). heterotopically, (in addition to the recipient s own organ) or
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Table 6. Success rates of RCA-transgenic porcine organs after transplantation to primate recipientsa
RCA Organ/kind of transplant Recipient Immuno-suppression Survival (days)
hDAF Heart/heterotropic Cynomologus þþþ , 135
hDAF Heterotopic Cynomologus þþ , 90
hDAF Orthopic Baboon þþþ , 28
hDAF Kidney/orthotopic Cynomologus þþ , 90
hCD59 Kidney/orthotopic, heterotopic Cynomologus þþþ , 20
hCD46 Heart, heterotopic Baboon þþ , 23
a
Abbreviations: þ, weak immunosuppression; þþ, moderate immunosuppression; þþþ, heavy immunosuppression; RCA, regulator of complement activity
orthotopically (life supportive) into non-human primates. that transgenic pigs will be available as organ donors
Survival rates reached 23 135 days with porcine xeno- within the next five to ten years. Guidelines for the clinical
grafts transgenic for one of the two complement regula- application of porcine xenotransplants are already avail-
tors; survival rates were heavily affected by the strength of able in the USA and are currently being developed in
the immune-suppressive protocol (Table 6) [29 31]. several other countries.
Similarly, transgenic expression of hCD59 was compatible The ethical challenges of xenotransplantation have
with an extended survival of porcine hearts in a perfusion been a matter of a worldwide intensive debate. A general
model or following transfer into primates [32,33]. These consensus has been reached that the technology is
data show that HAR can be overcome in a clinically ethically acceptable provided the individual well-being
acceptable manner by expressing human complement does not compromise public health by producing and
regulators in transgenic pigs [22]. transmitting new pathogens. Economically xenotrans-
Another promising strategy towards successful xeno- plantation might be viable if the enormous costs caused
transplantation is the knockout of the antigenic structures by patients suffering from severe kidney disease, needing
on the surface of the porcine organ that cause HAR. These dialysis or those suffering from chronic heart diseases
structures are known as 1,3-a-gal-epitopes and are could be avoided by a functional kidney or heart xenograft.
primarily produced by activity of 1,3-a-galactosyltransfer-
ase (a-gal). Piglets in which one allele of a-gal locus had Xenogenic cells and tissue
been knocked out by homologous recombination in Another promising area of application for transgenic
primary donor cells that were employed in nuclear animals will be the supply of xenogenic cells and tissue.
transfer were recently generated [34,35]. The birth of Several intractable diseases, disorders and injuries are
four healthy piglets with disruption of both allelic loci for associated with irreversible cell death and/or aberrant
a-gal has meanwhile also been published. Applying toxin A cellular function. Despite numerous attempts, primary
from Clostridium difficile to cells that already carried one human cells cannot yet be expanded well enough in
deleted a-gal allele selected a cell clone, which carried an culture. In the future, human embryonic stem cells could
inactivating point mutation on the second allele. This cell be a source for specific differentiated cell types that can be
clone was then used in nuclear transfer [36]. The used in cell therapy [48,49]. Xenogenic cells, in particular
usefulness of these animals for xenotransplantation has from the pig, hold great promise for successful cell
recently been reported [37]. therapies for human patients because cells can be
Further improvements in the success of xenotransplan- implanted at the optimal therapeutic location (i.e. immu-
tation will arise from the possibility of inducing a noprivileged sites, such as the brain), genetically or
permanent tolerance across xenogenic barriers [38,39]. A otherwise modified before transplantation to enhance
particularly promising strategy for long-term graft accep- cell function, banked and cryopreserved, or combined
tance is the induction of a permanent chimerism via with different cell types in the same graft [50].
intraportal injection of embryonic stem cells [40]. Xenogenic cell therapy has been advanced to preclinical
Prevention of transmission of zoonoses from the donor studies. Porcine islet cells have been transplanted to
animal to the human recipient is crucial for clinical diabetic patients and were shown to be at least partially
application of porcine xenografts. This aspect gained functional over a limited period of time [51]. Porcine fetal
particular significance when a few years ago it was neural cells were transplanted into the brain of
shown that porcine endogenous retroviruses (PERV) can patients suffering from Parkinson s and Huntington s
be produced by porcine cell lines and can even infect disease [52,53]. In a single autopsied patient the graft
human cell lines in vitro [41]. However, until today no survived for .7 months and the transplanted cells formed
infection has been found in patients that had received dopaminergic neurons and glial cells. Pig neurons
various forms of living porcine tissues (e.g. islet cells, extended axons from the graft site into the host brain
insulin, skin, extracorporal liver) for up to 12 years [42]. [52]. Further examples for the potential use of porcine
Recent intensive research has shown that porcine neural cells are in cases of stroke and focal epilepsy [54].
endogenous retroviruses probably do not present a risk Olfactory ensheathing cells (OECs) or Schwann cells
for recipients of xenotransplants provided all necessary derived from hCD59 transgenic pigs promoted axonal
precautions are taken [43 46]. In addition, a strain of regeneration in rat spinal cord lesion [55]. Thus, cells from
miniature pigs has been identified that does not produce genetically modified pigs could restore electrophysiologi-
infective PERV [47]. Although xenotransplantation poses cally functional axons across the site of a spinal cord
numerous further challenges to research, it is expected transsection. Xenogenic porcine cells could also be useful
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as a novel therapy for liver diseases. On transplantation of prion locus has been reported; however the cloned lambs
porcine hepatocytes to Watanabe heritable hyperlipidemic carrying the kockout locus died several days after birth
(WHHL) rabbits (a model for familial hypercholesterole- [65]. Prion knockout animals could be an appropriate
mia) the xenogenic cells migrated out of the vessels and model for studying the epidemiology of spongiform
integrated into the hepatic parenchyma. The integrated encephalopathies in humans and are crucial for
porcine hepatocytes provided functional low density developing strategies to eliminate prion carriers from a
lipoprotein (LDL) receptors and thus reduced cholesterol farm animal population.
levels by 30 60% for at least 100 days [56]. The pig could be a useful model to study defects of
A clone of bovine adrenocortical cells restored adrenal growth hormone releasing hormone (GHRH), which is
function upon transplantation to adrenalectomized severe characterized by a variety of conditions such as Turner
combined immunodeficient (SCID) mice indicating that syndrome, hypochrondroplasia, Crohn s disease, intra-
functional endocrine tissue can be derived from a single uterine growth retardation or renal insufficiency. Appli-
somatic cell [57]. Bovine neuronal cells were collected from cation of recombinant GHRH in an injectable form and its
transgenic fetuses, and when transplanted into the brain myogenic expression has been shown to alleviate these
of rats resulted in significant improvements in symptoms problems in a porcine model [66].
of Parkinson s disease [58]. Furthermore, xenotransplan- An important aspect of large animal models for human
tation of retinal pigment epithelial cells holds promise for diseases is the recent finding that somatic cloning per se
treating retinal diseases such as macular degeneration, does not result in shortening of the telomeres. Telomeres
which is associated with photoreceptor losses. Porcine or are highly repetitive DNA sequences at the end of the
bovine fetal cardiomyocytes or myoblasts might provide a chromosomes that are crucial for their structural integrity
therapeutic approach for the treatment of ischemic heart and function and are thought to be related to lifespan.
disease. Similarly, xenogenic porcine cells might be Telomere shortening is usually correlated with severe
valuable for the repair of skin or cartilage damage [50]. limitations of the regenerative capacity of cells, the onset
In light of the emergence of significantly improved of cancer, ageing and chronic disease with significant
protocols for genetic modification of donor animals and impact on human lifespan [67 70]. Expression of the
new powerful immunosuppressive drugs xenogenic cell enzyme telomerase, which is primarily responsible for the
therapy will evolve as an important therapeutic option for formation and rebuilding of telomeres, is suppressed in
the treatment of human diseases. most somatic tissues postnatally. Although telomeres in
cloned sheep (Dolly) derived from epithelial cells were
Farm animals as models for human diseases shorter than those of naturally bred age matched control
In mouse genetics the generation of knockout animals is a animals, telomere lengths in cloned mice and cattle were
standard procedure and several thousand strains carrying not different from those determined in age matched
gene knockouts or transgenes have been developed [Mouse controls even when senescent donor cells had been
Knockout and Mutation Database (MKMD); http:// employed [71 75]. Recent studies in our laboratory have
research.bmn.com/mkmd]. Models have been developed revealed that telomere length is established already early
for several human diseases. However, mouse physiology, in preimplantation development by a specific genetic
anatomy and life span differ significantly from those in programme and is dependent on telomerase activity [76].
humans, making the rodent model inappropriate for
several human diseases. Farm animals, such as pigs, Dietary modifications of animal products
sheep or even cattle could be more appropriate models to Application of gene and biotechnology for nutrition and
study human diseases in particular non-insulin-depen- biomedicine is more developed for plants than farm
dent diabetes, cancer and neurodegenerative disorders, animals [77]. The term nutriceuticals in the farm animal
which require longer observation periods than those context means that gene and biotechnology are used to
possible in mice [59 61]. With the aid of the microinjection enhance farm animal products to improve diet and have
technology an important pig model for the rare human eye concomitant medical applications. Because these products
disease Retinitis pigmentosa (PR) has been developed [62]. have a proven pharmacological effect on the body, they are
Patients with PR develop night blindness early in life regarded as  drugs and must be tested for efficacy and
attributed to a loss of photoreceptors. The transgenic pigs safety. Functional foods are those designed to provide
express a mutated rhodopsin gene and show a great specific and beneficial physiological effects on human
similarity with the human phenotype. Treatment models health and welfare and should prevent diet-related
with value for human patients are being developed [63]. diseases. Functional foods from animals could be used to
The development of the somatic cloning technology and lower cholesterol levels in an effort to battle cardiovascular
the merger with targeted genetic modifications and diseases, to reduce high blood pressure by adding
conditional gene expression will enhance the possibilities angiotensin converting enzyme (ACE) inhibitors or to
for creating useful models for human diseases in large increase immunity by adding specific immunostimulatory
animals. A good example is the knockout of the prion gene peptides [78]. However, data showing that nutriceuticals
that would make sheep and cattle non-susceptible to are really beneficial for human health are rare.
spongiform encephalopathies (scrapie and BSE). Mice Regarding the production of improved quality animal
models showed that the knockout of the prion protein is the products, interesting observations have been made in
only secure way to prevent infection and transmission of several beef cattle breeds, such as Belgian Blue and
the disease [64]. The first successful targeting of the ovine Piedmontese. These breeds were accidentally bred for
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Review TRENDS in Biotechnology Vol.22 No.6 June 2004
mutations of the myostatin gene, which renders it non- environmental pollution. These pigs carry a bacterial
functional or less functional than the wild-type gene [79]. phytase gene under the transcriptional control of a
The mutated genes cause muscle hypertrophy and led to salivary gland specific promoter, which allows the pigs to
improved meat quality. This observation makes targeted
digest plant phytate. Without the bacterial enzyme, the
modifications of the myostatin gene an interesting option
phytate phosphorus passes undigested into manure and
for the meat industry. A diet rich in non-saturated fatty
pollutes the environment. With the bacterial enzyme, the
acids is correlated with a reduced risk of stroke and
fecal phosphorus output was reduced up to 75% [86]. These
coronary diseases. One transgenic approach to this is the
environmentally friendly pigs are expected to enter the
generation of pigs with increased amounts of non-
commercial production chains within the next few years.
saturated fatty acids. Pigs producing a higher ratio of
unsaturated versus saturated fatty acids in their muscles
Precautions and perspectives
are currently under development in Japan.
Throughout mankind s history farm animals have made
An attractive example for targeted genetic modification
significant contributions to human health and well-being.
could be dairy production [80,81]. Apart from conventional
The convergence of the recent advances in reproductive
dairy products, it could be possible to produce fat-reduced
technologies with the tools of molecular biology opens a
or even fat-free milk or milk with a modified lipid
new dimension for this area [1]. Major prerequisites will be
composition via modulation of enzymes involved in lipid
the continuous refinement of reproductive biotechnologies
metabolism; to increase curd and cheese production by
and a rapid completion of livestock genome sequencing
enhancing expression of the casein gene family in the
and annotation. The technology developed in the decipher-
mammary gland; to create  hypoallergenic milk by knock-
ing of the human genome will improve and accelerate
out of the b-lactoglobulin gene; to generate lactose-free
sequencing of genomes from livestock [87]. We anticipate
milk via knockout of the a-lactalbumin locus that is the
genetically modified animals will play a significant role in
key molecule in milk sugar synthesis; to produce  infant
the biomedical arena, in particular via the production of
milk in which human lactoferrin is abundantly available
valuable pharmaceutical proteins and the derivation of
or to produce milk with a highly improved hygienic
xenografts, within the next 5 7 years. Agricultural
standard via an increased level of lysozyme or other
application might be further away (.10 years) given the
anti-microbiological substances in the udder. Lactose-
complexity of some of the economically important traits
reduced or lactose-free milk could make dairy products
and the public skepticism of genetic modification related to
suitable for consumption by a large proportion of the
food production [1].
world s population who do not possess an active lactase
A crucial aspect of animal-derived products is the
enzyme in their gut system. However, one has to bear in
prevention of transmission of pathogens from animals to
mind that lactose is the main osmotically active substance
humans. This requires sensitive and reliable diagnostic
in milk and a lack thereof could interfere with milk
and screening methods for the various types of pathogenic
secretion. A lactase construct has been expressed in the
organisms. The recent findings (see above) that the risk of
mammary gland of transgenic mice and reduced lactose
PERV transmission is negligible are promising and show
contents by 50 85% without altering milk secretion [82].
that with targeted and intensive research such important
However, mice with a homozygous knockout for a-
questions can be answered within a limited period of time,
lactalbumin could not feed their offspring because of the
paving the way for preclinical testing of xenografts.
high viscosity of the milk [83]. These diverging findings
Furthermore, it should be kept in mind that the biomedical
demonstrate the feasibility of obtaining significant altera-
applications of farm animals will require strict standards
tions of milk composition by applying the appropriate
of  genetic security and reliable and sensitive methods for
strategy.
the molecular characterization of the products. A major
The physicochemical properties of milk are mainly
contribution towards the goal of well-defined products will
affected by the ratio of casein variants. Therefore, casein is
come from array technology (cDNA, peptide or protein
a prime target for the improvement of milk composition.
arrays), which establishes  fingerprint profiles at the
Mouse models have been developed for most of the above
transcriptional and/or protein level [88,89]. Meanwhile
modifications indicating the feasibility of obtaining sig-
improvements of RNA isolation and unbiased amplifica-
nificant alterations in milk composition but at the same
tion of tiny amounts of mRNA (picogram) enable research-
time showing that unwanted side effects cannot be ruled
ers to analyse RNA from single embryos [90]. With the aid
out [83,84]. Only one full-scale study in livestock has been
of this technology one can gain in-depth insight into the
reported as yet [85]. The recent report showed that the
proper functioning of a transgenic organism and thereby
casein ratio can be altered by overexpression of b- and
ensure the absence of unwanted side effects [88,89]. This
k-casein in cattle clearly underpinning the potential for
would also be required to maintain the highest possible
improvements in the functional properties of bovine
levels of animal welfare in cases of genetic modification.
milk [85].
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