Ž . Ž
. knockout
Capecchi, 1989; Kilby et al., 1993 . In light of the recent advances, somatic nuclear transfer holds the greatest promise for significant improvements in the genera-
tion of transgenic livestock. A major prerequisite is the availability of suitable cell lines compatible with techniques for precise genetic modifications either for gain or loss of
function. Another premise is a significantly improved knowledge of gene sequences and organization of the livestock genome, which currently is lagging much behind that of
mouse and human, where the putative 3 billion bp are expected to be sequenced by the year 2003. This review focuses on recent achievements in transgenic livestock as
generated via microinjection and briefly outlines the potential for improving transgenic technology in livestock species by nuclear transfer and application of sophisticated
molecular tools.
2. Current technology — application of microinjection
The above limitations of microinjection technology have restricted the number of studies in this area. While thousands of scientific articles have been published dealing
Ž with transgenic andror knockout mice e.g. mouse knockout and mutation database
. Ž
www.bimedvet.comrdatabasesrcurrbiolrMkordataset.exe , significantly fewer -
. Ž
400 reports are known for livestock from which almost a quarter are reviews Wall, 1996, Sinai’s mammary transgene database: http:rrmbcr.bcm.tmc.edu: 80rBEPr
. ERMBrmtdbrhtml . Attributed to the enormous amounts of resources needed for
transgenic livestock production, the costs for one expressing transgenic animal are extraordinary high. It has been calculated that one expressing transgenic mouse requires
average expenses of US120 whereas one expressing transgenic pig would amount to US25,000, one transgenic sheep US60,000 and one transgenic cow US546,000 when
Ž .
in vivo derived zygotes are used Wall et al., 1992 . Transgenic production in cattle can only be practical through in vitro production of embryos as it reduces costs by 50–60.
From a total of more than 36,500 microinjected zygotes f 2300 developed to blasto- cysts, upon transfer 28 resulted in pregnancy and 18 transgenic calves could be
identified. To improve efficiency of the procedure the embryos were biopsied and
Ž .
analyzed by PCR for the presence of the transgene Eyestone, 1999 . The early detection Ž
of transgenesis in preimplantation embryos has been shown to be feasible Bowen et al., .
1994; Hyttinen et al., 1994 ; however, efficiency is limited due to an early onset of Ž
. mosaicism Lemme et al., 1994 . The propagation of the transgenic trait in a given cattle
population can be accomplished through in vitro production techniques by using semen from a transgenic bull for in vitro fertilization and collecting oocytes by means of
ultrasound-guided follicular aspiration from transgenic female founder animals and their
Ž .
subsequent use in IVF Eyestone, 1999 . Details of the microinjection technology and Ž
the potential applications of transgenic livestock have been extensively reviewed Re- xroad, 1992; Pursel and Rexroad, 1993; Niemann et al., 1994; Wall, 1996; Wall et al.,
. 1992; Murray, 1999 . Despite the inherent limitations, microinjection has allowed
commercial exploitation of transgenic animals for biomedical purposes and even for a few agricultural traits.
2.1. Transgenic animals with agricultural traits An Australian group has generated transgenic pigs bearing a modified porcine growth
Ž .
hormone hMt-pGH
construct that can tightly be regulated by zinc feeding. The transgenic animals show significant improvements in economically important traits such
as growth rate, feed conversion and body fat muscle ratio. These animals are close to Ž
. being released to the market
Nottle et al., 1999 . Transgenic sheep carrying a keratin-IGF-I construct show expression in the skin and the clear fleece was about 6.2
greater in transgenic vs. nontransgenic animals. These animals are also being prepared Ž
. for commercial application Damak et al., 1996a,b . In both projects, no adverse effects
of the transgene on health or reproduction were observed. Another interesting applica- Ž
. tion could be enhanced disease resistance Muller and Brem, 1994 . Recently, a mouse
¨
model was established in which recombinant monoclonal antibodies, which neutralize Ž
. the transmissible gastroenteritis virus
TGV , are secreted into milk and provided Ž
. passive protection against gastroenteric infections to the pups Castilla et al., 1998 . The
verification of this model in pigs is promising. 2.2. Transgenic animals in biomedicine
Transgenic technology is well advanced in biomedicine. Several recombinant proteins have been produced in large amounts in the mammary gland of transgenic sheep and
Ž goats, purified from milk and biologically characterized Houdebine, 1994; Meade et al.,
. Ž
. 1999 . Several products such as human antithrombin III ATIII , a -antitrypsin, tissue
1
Ž .
plasminogen activator tPA , a-glucosidase and lactoferrin are currently in advanced clinical trials and the first product is expected to be on the market at the beginning of the
Ž .
next century Ziomek, 1998; Meade et al., 1999 . Although a variety of proteins have been efficiently produced in the mammary gland of transgenic animals, not every protein
can obviously be produced at the desired high amounts. Cattle transgenic for human Ž
. erythropoetin hEPO have been described, but expression in milk has not been shown
Ž .
Hyttinen et al., 1994 . Ectopic hEPO-expression in organs other than the mammary Ž
gland was shown to be associated with premature death of transgenic rabbits Massoud .
et al., 1996 . We have demonstrated that human clotting factor VIII cDNA constructs can be expressed in the mammary gland of transgenic sheep. However, the recovery
rates of hFVIII protein were low and dependent on the individual donor, storage temperature and dilution of milk samples. hFVIII was rapidly sequestered in ovine milk
Ž
. Halter et al., 1993; Niemann et al., 1994, 1999b; Guzik and Niemann, 1995 . From
; 1500 transferred microinjected zygotes 14 transgenic offspring were obtained al-
Ž .
though the pregnancy rate was remarkably high with 63 Table 2 . Interestingly, transgenic offspring were only obtained with constructs bearing the ovine b-lacto-
globulin promoter element and not with constructs bearing the WAP promoter. In previous experiments, we had shown that the WAP-hFVIII constructs were compatible
Ž .
with survival and normal development of transgenic mice Espanion et al., 1997 . Our transgenic sheep had similar birth weights and showed similar development as the
nontransgenic controls. In current experiments, we aim at increasing expression levels
Table 2 Efficiency of microinjection of hFVIII cDNA constructs into ovine pronuclei
Data are based on experiments from 10 breeding seasons, hFVIII constructs: b-lac-hFVIII; b-lac-hFVIII-Mt-I, Ž
. MAR-b-lac-hFVIII-MT-I for details see Halter et al., 1993; Niemann et al., 1999b .
n Microinjected zygotes
1948 –
Lysis 265
13.7 Transferred zygotes
1447 –
Recipients 569
– Pregnancies
356 62.6
Lambs born 543
– Lambsrrecipient
0.95 –
Lambsrpregnancy 1.5
– Lambsrtransferred zygote
– 37.5
Transgenics 14
– Transgenicsrtransferred zygote
– 1.0
Transgenicsrlambs born –
2.6
by employing novel constructs that include the genomic DNA of the extraordinary large and complex regulated hFVIII gene.
Xenotransplantation is another promising area in which transgenic pigs are close to clinical application. To overcome the growing shortage of human organs, transgenic pigs
have been generated that express human complement regulatory genes. This approach Ž
. enables overcoming the hyperacute rejection response HAR as shown by an average
survival rate of 40–90 days of immunosuppressed primates having received a heart from Ž
. Ž
an hDAF decay accelerating factor transgenic pig Cozzi and White, 1995; White, .
1996; Platt and Lin, 1998 . The complement regulatory protein hCD59 interferes with Ž
. the formation of the membrane attack complex MAC at the end of the complement
cascade. We have microinjected appropriate hCD59 constructs into pronuclei of porcine zygotes. To improve the yields of transgenics we have modified several methodological
Ž details of the procedure. A higher DNA concentration 8–10 ngrml instead of 4–5
. Ž
ngrml in the injection buffer increased the number of transgenic offspring 15 vs. .
Ž .
5 and confirmed recent results
Nottle et al., 1997 . After transfer of 20–30 microinjected zygotes per recipient, the percentage of transgenic offspring was 16–20,
which decreased to 8 when more than 30 zygotes had been transferred, although in the Ž
. latter recipients, the pregnancy rates were considerably higher 80 vs. 50 . Transfer
of the microinjected zygotes into one oviduct was compatible with high pregnancy rates and acceptable litter sizes. We have recently identified transgenic pigs that show high
expression of hCD59 predominantly in the heart, but also other target organs. Further- more, transgenic endothelial cells and fibroblasts were protected against complement
Ž mediated lysis showing that the human CD59 is biologically active Niemann et al.,
. 1999a and manuscript in preparation . Although xenotransplantation poses numerous
other challenges to research, it is expected that transgenic pigs will be available as organ donors within the next 5–10 years. One major prerequisite is the prevention of the
potential transmission of pathogenic microorganisms, in particular porcine endogenous
Ž .
retroviruses PERV . Another promising area of application for transgenic animals will be tissue engineering. Recently, neuronal cells were collected from bovine transgenic
fetuses, transplanted into the brain of a rat model for Parkinson disease and resulted in Ž
. significant improvements of the neurological symptoms Zawada et al., 1998 . This
indicates that genetically modified livestock cells may serve as a suitable source for xenogenous tissue in certain diseases.
The above brief description demonstrates that although the requirements to generate a transgenic animal that efficiently expresses its transgene are enormous; within less than
20 years transgenic livestock have emerged that will provide valuable contributions to human health. With increasing knowledge of the genetic basis of agricultural traits and
improvements in the technology to generate transgenic animals, numerous further commercial applications are expected to be developed. The use of transgenic farm
animals for biomedical applications in particular as organ donors for xenotransplantation
Ž .
or as appropriate disease models Petters et al., 1997; Theuring et al., 1997 will require precise genetic modifications and a tight control of transgene expression.
3. Control of transgene expression
