www.elsevier.comrlocateranireprosci
Transgenic livestock: premises and promises
H. Niemann
), W.A. Kues
( )
Department of Biotechnology, Institut fur Tierzucht und Tier¨ Õerhalten FAL , Mariensee,
31535 Neustadt, Germany
Abstract
Microinjection of DNA constructs into pronuclei of zygotes has been the method of choice for the generation of transgenic livestock. However, this procedure is characterized by low efficiency
Ž1–4% transgenic offspring , random integration and variable expression of the transgene as well.
as a considerable proportion of mosaicism. Furthermore, it is extremely time consuming and costly. As a consequence, commercial application has focused on the production of recombinant proteins in the mammary gland of transgenic animals and xenotransplantation, e.g. the use of porcine organs in human organ transplantation. In addition, transgenic pigs carrying a modified
Ž .
porcine growth hormone hMt-pGH construct show significant improvements in economically important traits without adverse side effects of a GH overproduction. Improvements of transgenic technology will likely come from the generation of appropriate cell lines suitable for transfection or even homologous recombination and their subsequent use in nuclear transfer. Additionally, in the mouse a number of sophisticated molecular tools have been developed that allow precise modifications of the genome. These include the application of artificial chromosomes from yeast
ŽYAC or bacteria BAC for position-independent and copy-number-dependent expression of a. Ž .
Ž .
transgene, the Tet-system tetracycline inducible for a tight temporal control of transgene
Ž
expression, as well as conditional mutagenesis by applying site-specific DNA recombinases e.g.
.
Cre, FLP . The successful adaptation of these molecular tools to livestock will enable the fulfilment of many of the promises originally thought to be achievable when transgenic livestock were first reported.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Livestock; Transgenic; Homologous recombination; Artificial chromosomes; Nuclear transfer;
Conditional mutagenesis
)Corresponding author. Tel.:q49-5034-871-148; fax:q49-5034-871-101.
Ž .
E-mail address: niemann@tzv.fal.de H. Niemann .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
1. Introduction
Microinjection of foreign DNA into pronuclei of fertilized oocytes has been the only
Ž .
successful method for the generation of transgenic livestock Table 1 . Although this
Ž
procedure works reliably, it is inefficient 1–4% transgenic offspringrtransferred
mi-.
croinjected zygotes , results in random integration into the host genome and variable
Ž .
expression due to position effects Pursel and Rexroad, 1993; Wall, 1996 . In addition, it is time consuming and requires substantial intellectual, financial and material resources
ŽSeidel, 1993 . As a consequence, research has focused on alternate methodologies for.
improving the generation of transgenic livestock. These include sperm mediated DNA
Ž . Ž .
transfer Gandolfi, 1998; Squires, 1999 , the intracytoplasmic injection ICSI of
trans-Ž .
genic sperm heads Perry et al., 1999 , the use of retroviral vectors either by injection or
Ž .
infection of oocytes or embryos Haskell and Bowen, 1995; Chan et al., 1998 or the use
Ž
of genetically modified ovine, bovine or caprine donor cells in nuclear transfer Schnieke
.
et al., 1997; Cibelli et al., 1998a; Baguisi et al., 1999 . Further improvements may be derived from the adaptation of technologies that are effective in allowing precise genomic modifications of the murine genome. These include targeted chromosomal integration by DNA recombinases, such as Cre or FLP or homologous recombination that would enable to generate transgenic animals with a gain or a loss of function
Table 1
Methodological repertoire for the production of genetically modified mice and large farm animals
q sshown;y snot shown; ?squestionable; approaches were validated according to germline transmission and expression data.
Methodology Integration Expression Mouse Livestock
Gain-of-function
Microinjection
minigene constructs random variable q q
Ž .
with regulatable promoter random inducible MtrTET qrq qry
artificial chromosomes random integration site independent q y ŽYAC, BAC.
Retroviral infection randomrsite variable? q q?
specific
Ž .
Sperm atogonia mediated random ? q q?
Targeted chromosomal integration defined controlled q y
ŽFlp, Crerlox.
a
Nuclear transfer with transgenic randomrdefined variabler? y qrq donor cells
Loss-of-function
ES cellsqgene knockout defined abolished in all cells q y
Cell-type-specific knockout defined abolished in specific cells q y
Inducible knockout defined abolished upon induction q y
Somatic nuclear transfer with defined abolished y? y?
knockout cells
a Ž .
Žknockout. ŽCapecchi, 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 US$120 whereas one expressing transgenic pig would amount to US$25,000, one transgenic sheep US$60,000 and one transgenic cow US$546,000 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 f2300 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 ReRe-xroad, 1993; Niemann et al., 1994; Wall, 1996; Wall et al.,
.
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 , a1-antitrypsin, tissue
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
ŽHyttinen 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
ŽHalter 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 .
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 %
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.,
.
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
3.1. Inducible gene expression
A major progress in current transgenic technology would be a tight control of transgene expression. Control elements that are known to regulate the activity of
Ž .
transgenes are the metallothionein promoter Nottle et al., 1999 , heat shock promoter, or steroid responsive elements. However, these have been used with limited success attributed to low induction levels and physiological effects of the inducer elements
ŽYarranton, 1992 . Significant improvements of the temporal control of gene expression. Ž
could be achieved by employing the tetracycline regulatable system Gossen et al.,
.
1995 . This involves a transcriptional transactivator, which has been created by fusion of the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires the presence of a tetracycline analogue for DNA binding and transcriptional activation. It has been shown that the presence of a tetracycline analogue
Ž . Ž
led to a burst of expression in cell lines and even transgenic mice Tet-on Furth et al.,
.
1994; Gossen et al., 1995 . This system can also be modified in a way that the presence
Ž .
of tetracycline suppresses expression of the target gene Tet-off . In this system, tetracycline binds to the transactivator, blocks transcription activity and shuts down
Ž .
expression of the desired transgene Furth et al., 1994; Mayford et al., 1995 . The original technology requires two independent integration sites, e.g. two different lines of transgenic mice which are subsequently bred to obtain a transgenic animal harbouring both transgenic modifications. This makes it unfeasible to introduce the Tet-system into livestock. However, recent development indicates that both control elements can be integrated in a single plasmid and allow efficient and tight control of gene expression in
Ž .
vitro and in vivo Schultze et al., 1996 . Gene expression could be depressed by administration of tetracycline in transgenic mice and removal of the antibiotic induced
Ž .
( )
3.2. Internal ribosome entry sites IRES and dicistronic RNAs
Another possibility to improve transgene expression is to combine two or even more
Ž
transgenes by employing IRES elements Mountfort and Smith, 1995; Houdebine and
.
Attal, 1999 . The function of IRES has been demonstrated in poliovirus. In this system, the translation of the downstream cistron is regulated independently by direct association
Ž
of ribosomes with the IRES, e.g. these act as ribosome landing pad Pelletier and
. X
Sonenberg, 1988 . IRES elements are naturally occurring in the 5 UTR sequences of viruses and cells. They can be employed to create artificial dicistronic or even
Ž .
multicistronic cassettes Mountford and Smith, 1995 . An attractive application would be to couple expression of the target gene with that of a suitable reporter such as GFP
Žgreen fluorescent protein . Such approach would facilitate detection of expression in a.
significant manner. Appropriate dicistronic constructs can be microinjected into porcine
Ž .
pronuclei and are compatible with normal development unpublished own observation .
3.3. Artificial chromosomes
It has been shown that the control of transgene expression can be improved by increasing the length of the flanking genomic DNA sequences. Artificial chromosomes
Ž .
are able to carry extremely large DNA fragments of more than one megabase Mb . Artificial chromosomes have been invented from yeast; they include centromeres, telomeres, and origins of replication as essential components. Microinjection of a 450-kb genomic YAC harbouring the murine tyrosinase gene resulted in transgenic mice, which showed a position-independent and copy-number-dependent expression of the transgene.
Ž
Albinism was rescued in transgenic mice and rabbits Schedl et al., 1992, 1993; Brem et
.
al., 1996 . A 210-kb YAC construct has been microinjected into rat pronuclei and
a-lactoglobulin and human growth factor were expressed in the mammary gland of
Ž .
transgenic rats Fujiwara et al., 1997, 1999 . Up to now, transgenic livestock have not been reported with YAC constructs. This may be attributed to the inherent problems of this technology, such as difficulties to isolate YAC DNA with sufficient purity and the
Ž
enormous instability with a tendency for deleting regions from the insert Monaco and
. Ž .
Larin, 1994 . Artificial chromosomes can also be constructed in bacteria BACs , which can easily be genetically modified and even allow homologous recombination. Trans-genic mice were generated via pronuclear injection of BACs and germline transmission
Ž .
and proper expression of the transgene was achieved Yang et al., 1997 . Recently, also
Ž .
mammalian artificial chromosomes MAC have been engineered by employing endoge-nous chromosomal elements from YACs or extra chromosomal elements from viruses or
Ž . Ž .
BACs and P1 artificial chromosomes PACs Vos, 1997 . MACs with a size of 1–5 Mb containing YAC telomeres were formed by de novo mechanism; they segregated like
Ž .
4. Future technology — marriage of nuclear transfer and advanced molecular tools
Ž
Recent reports on the generation of transgenic sheep and cattle Schnieke et al., 1997;
.
Cibelli et al., 1998b via somatic nuclear transfer inspired great expectations about this elegant approach to improve the generation of transgenic livestock. Fetal fibroblasts
were transfected in vitro, screened for transgene integration and then transferred into enucleated oocytes. After fusion of both components and activation of the reconstituted nuclear transfer complexes, blastocysts were transfered to synchronized recipients and
Ž .
gave rise to transgenic offspring Fig. 1 . Compared with the microinjection procedure in which screening for transgenesis and optimal expression of the transgene takes place at the level of the offspring, cloning by nuclear transfer can accelerate the time-consum-ing transgenic production by prescreentime-consum-ing of donor cells for the optimal expression of the desired trait in vitro and 100% transgenic offspring.
As of today a variety of cell types has been successfully employed as donors in
Ž .
nuclear transfer Fulka et al., 1998 . However, the overall efficiency of nuclear transfer is low. Factors affecting the success of nuclear transfer are poorly defined and the percentage of live offspring does not exceed 1–3% of the transferred reconstituted
Ž .
embryos Cibelli et al., 1998b; Wilmut et al., 1997; Wakayama et al., 1998 . A better understanding of the underlying fundamental molecular processes, such as cell cycle
Ž .
compatibilities between cytoplasm and donor nucleus Campbell et al., 1996 , cell cycle
Ž .
synchronization of the donor cells Boquest et al., 1999; Kues et al., 2000 , reprogram-ming and the relevance of differentiation vs. totipotency is urgently needed. Upon serum deprivation or treatment with chemical cell cycle inhibitors, the majority of porcine donor cells was synchronized at the presumptive optimal cell cycle stage at G0rG1
Ž .
without compromising their viability Kues et al., 2000 . This contributes substantially to standardize the nuclear transfer procedure as much as possible. In addition, methods have to be established that allow reliable determination of the capacity of a given nuclear transfer embryo to develop into a normal offspring. Currently, an increased peri-and postnatal mortality is found in offspring derived from nuclear transfer embryos
ŽWilmut et al., 1997; Kato et al., 1998 ..
( )
4.1. Permanent cell lines ES, EG, EC
The basis for loss of function transgenics in the mouse are the availability of
Ž .
embryonic stem cells ES cells , molecular tools for homologous recombination and the high probability after injection into host blastocysts with which ES cells give rise to
Ž .
germline contribution Evans and Kaufman, 1981; Martin, 1981 . This provides a powerful approach to introduce specific genetic changes into the murine genome. The essential characteristics of ES cells include derivation from the preimplantation embryo
Žinner cell mass cells , undifferentiated proliferation in vitro and the developmental.
potential to differentiate into all cell types. Morphological markers for ES cells are the growth in three dimensional colonies, formation of embryoid bodies in vitro and of
Ž .
teratocarcinomas upon transplantation in immunodeficient mice Hogan et al., 1994 . Molecular markers used for murine ES cells are the stage-specific embryonic antigen-1
ŽSSEA-1 , SSEA-3, SSEA-4, TA-60-1, octamer-binding transcription factor-4 Oct-4 ,. Ž .
alcaline phosphatase activity, high telomerase activity as well as a lack of differentiation
Ž .
markers Hogan et al., 1994 .
Ž . Ž .
Ž . Ž .
germ cells PGC Matsui et al., 1992; Resnick et al., 1992 and share several characteristics with ES cells, including morphology, pluripotency, and the capacity for germline transmission. Similar to ES cells, EG cells express alkaline phosphatase and Oct-4. They can be aggregated to form embryoid bodies and give rise to teratocarcino-mas when transferred to appropriate ectopic sites. However, up to now, homologous recombination has not been achieved in this cell type. Murine EC cells were originally
Ž .
derived from induced teratocarcinomas Hogan et al., 1994 . However, they differentiate only rarely into gamete cells of reconstituted chimeras and are therefore not applicable for transgenic animal production.
Thus, the ultimate criterion for true totipotent stem cell lines is the contribution to the germline either in chimeras or by starting a new development upon nuclear transfer.
ES-Ž
and EG-like cell lines have been isolated from sheep, pig and cattle Wheeler, 1994;
.
Anderson, 1999 . Porcine and bovine cell lines were capable of contributing to chimera
Ž
formation upon injection into appropriate host blastocysts Wheeler, 1994; Shim et al.,
.
1997; Cibelli et al., 1998a; Piedrahita el al., 1998 . However, no germline transmission has been reported so far. True totipotent stem cell lines might require specific culture conditions, growth factor supplements and probably a specific genetic background, as
Ž .
even only few mouse strains are suitable for ES cell isolation Hogan et al., 1994 .
4.2. Homologous recombination in liÕestock
Homologous recombination in murine ES cells is the most direct and unambiguous way to eliminate gene function and is therefore the preferred method to establish a null genotype. Several strategies for gene targeting in murine ES cells have been developed
ŽMayford et al., 1995; Kuhn and Schwenk, 1997; Muller, 1999 . More than 1000
¨
¨
. Žknockout strains have been created via gene targeting in ES cells mouse knockout and
.
mutation database www.biomednet.com . Prominent examples of gene knockouts are related to certain oncogenes, kinases, phosphatases, growth factors, transcription factors
Ž
and disease associated genes Bunz et al., 1998; Shastry, 1998; Gotz et al., 1998; Muller,
¨
¨
.1999 . The potential for a gene knockout technique in livestock production is high-lighted by the discovery that several beef cattle breeds, like Belgian Blue and Piedmon-tese, are accidentally homozygous for a mutated myostatin gene, which is functionally
Ž
inactive and could be referred to as a natural knockout Grobet et al., 1997; Kambadur et
.
al., 1997; McPherron and Lee, 1997 . The similarity in phenotypes of myostatin mutated
Ž .
cattle and myostatin null mice McPherron et al., 1997 is striking and suggests that myostatin is a potentially useful target for genetic modification in farm animals.
Gene targeting in farm animals is hampered by the lack of true totipotent stem cells. However, the definition of totipotency has to be reconsidered, since the successful
Ž
cloning of sheep and cattle from adult mammary epithelial cells and fibroblasts Wilmut
.
et al., 1997; Cibelli et al., 1998b . Nuclear transfer techniques promise to circumvent the need for true totipotent cells for the generation of loss-of-function transgenic livestock. The future challenge in transgenic farm animal production is the isolation and handling
Ž
of primary cell cultures, either from somatic or embryonic origin Schnieke et al., 1997;
.
modifications, clonal selection and subsequently for nuclear transfer. Gene targeting in somatic cells of livestock will have important applications in combination with nuclear transfer. Although gene targeting in somatic cells is relatively uncommon, recent
Ž . Ž
progress in rat cells Mateyak et al., 1997 and non-immortalized human cells Brown et
.
al., 1997; Bunz et al., 1998 clearly shows the significant potential of this field. Moreover, isogenic DNA does not seem to be a prerequisite for efficient gene targeting
Ž .
in human somatic cells Sedivy and Dutriaux, 1999 . Recently, Pharmaceutical Proteins
Ž .
Limited PPL has announced the birth of lambs produced by nuclear transfer of gene
Ž
targeted somatic cells, but no details have been disclosed Transgenic Animal Research
.
Conference, August 1999, Lake Tahoe, CA, USA .
Gene targeting in somatic cells has been difficult to achieve because the absolute frequency of homologous recombination events in somatic cells is two orders of
Ž
magnitude lower than in ES cells Arbones et al., 1994; Hanson and Sedivy, 1995;
. Ž .
Brown et al., 1997 and the frequency of nonhomologous illegitimate recombination events is typically high. Targeting constructs must provide an efficient enrichment of homologously over nonhomologously recombined clones. The classical positive–nega-tive strategy uses vectors that are based on a negapositive–nega-tively selectable gene which is placed on the flanks of the targeting sequences, and which is removed in the homologous recombination process. Additionally, an independent expression cassette for positive selection is needed. In contrast, in a promoterless targeting strategy the positively selectable marker lacks a suitable promoter and is driven by the endogenous promoter of the target gene after homologous recombination. Nonhomologous recombination events by random integration close to a chromosomal promoter seem to be relative rare
ŽHanson and Sedivy, 1995 . These techniques promise to combine gene targeting in.
somatic cells and nuclear transfer for progress in transgenic large animal production.
4.3. Conditional mutagenesis and region specific-knockouts
The above findings demonstrate the power of gene targeting, but they also point to a need for additional molecular techniques when precise genetic modifications are desired. Gene knockouts per se have no spatial or temporal restriction. As the targeted gene product is absent for the entire life of the animal in all cells, it is difficult to assign a phenotype to a specific knockout as a mutant organism may compensate for the loss of a gene product or the knockout may have complex, secondary effects, e.g. depending on
Ž .
the genetic background Gerlai, 1996 , or may even result in embryonic lethality. A powerful tool for the design of genetic switches and for accelerating the creation of genetically modified animals, is the Cre site-specific DNA recombinase of bacteriophage
Ž .
P1 Sauer, 1998 . A single 38-kDa Cre protein is required to catalyze recombination between two loxP recognition sites, consisting of 34-bp DNA sequences. Recombination can occur between directly repeated loxP sites on the same molecule to excise the intervening DNA sequence, irrespective of whether the recognition sites are located on a
Ž .
plasmid or a mammalian chromosome Sauer and Henderson, 1988 . The combination of tissue-specific promoter elements with the Cre DNA recombinase enables restriction
Ž .
Two mouse strains are required for this approach. The first is a conventional transgenic strain in which the DNA recombinase is expressed in a cell-type-restricted manner. The second strain is generated by using gene targeting in ES cells. The second strain carries the target gene flanked on either end by loxP sequences. A crucial caveat is that the insertion of these loxP sites must not disrupt the normal function of the target gene. The two genetic alterations are brought together in one mouse through mating. This will give rise to a line of mice in which the target gene is deleted only in those cells that express the recombinase. Deletion of the DNA polymeraseb gene in the germline resulted in a lethal phenotype, but deletion of this gene in T cells gave rise to viable animals in which
Ž .
the effects of the knockout in a specific cell type can be analyzed Gu et al., 1994 . Moreover, employing an inducible promoter, like the interferon-responsive promoter to control the expression of Cre recombinase allows insertion of an inducible gene
Ž .
knockout Kuhn et al., 1995 . In principle, recombinase-mediated recombination should
¨
also allow gene replacement, e.g. the exchange of the reading frames of milk protein genes with the sequences of genes encoding pharmaceutical proteins.
5. Conclusions and outlook
The combination of recent advancements in reproductive technologies with the tools of molecular biology opens the horizon to a new era in transgenic biotechnology. Most of the described methods and strategies are currently applicable only in the mouse model and their adaptation to generate transgenic livestock has to be shown. Tissue specific expression, inducible gene knockouts or knockins, targeted chromosomal integration or artificial chromosomes are challenges for research even in mouse genetics and are far from being standard methodology. Thus, substantial progress has to be made in the livestock sector to reach the advanced methodological stage and the status of sequencing already available for the mouse. The growing amount of data from the human genome project will certainly inspire intense genome sequencing in livestock and will yield valuable information on genomic structure and function. To handle this boost of genetic information will require suitable tools in bioinformatics. The exciting developments in somatic cloning pave the way for the introduction of loss-of-function transgenics in livestock. Major prerequisites are an optimized nuclear transfer protocol and substantial progress in the handling of somatic cells. However, it should be kept in mind that putative biomedical applications such as xenotransplantation or usage of transgenic farm animals for food production will require strict standards on ‘‘genetic security’’ and reliable and sensitive methods for the molecular characterization of the ‘‘products’’. A major contribution towards this goal will come from DNA chips or arrays establishing profiles at the transcriptional andror protein level and allowing in-depth insight into the proper function of a transgenic organism. Besides the technical problems, public acceptance of recombinant products has to be taken into account. Additionally, welfare of the transgenic animals must be secured and pain or suffering due to genetic
Ž .
Acknowledgements
The authors express their sincere gratitude to Christa Knochelmann for the careful
¨
preparation of this manuscript. We thank Dr. Christine Wrenzycki for providing Fig. 1. The research on which this review is based was funded by grants from the Deutsche
Ž .
Forschungsgemeinschaft DFGSFB 265 and the Federal Ministry for Research and
Ž .
Development BMBF .
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