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
Ž .
Yarranton, 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 .
expression of a reporter gene construct by more than 800-fold Schultze et al., 1996 .
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
Ž .
green 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 .
normal chromosomes upon introduction into cell lines Ikeno et al., 1998 . It can be anticipated that by the recent development of MACs, limitations of YACs or BACs
might be overcome and transgenic livestock could be created harbouring the genomic elements of a desired gene or gene cluster. In addition, such artificial chromosomes may
also be introduced into appropriate cell lines that can be transferred to enucleated oocytes in the process called ‘‘nuclear transfer’’.
4. Future technology — marriage of nuclear transfer and advanced molecular tools
