Recent Progress of Transgenic
Pig Models for Biomedicine and
Pharmaceutical Research
Authors: Wiebke Garrels, Heiner Niemann.
Corresponding author: Wilfried A. Kues
Abstract
The first transgenic pigs were produced by the microinjection of
foreign DNA into zygotic pronuclei in 1985. Since then, the methodological repertoire for porcine transgenesis was expanded to
somatic cell nuclear transfer, lentiviral transgenesis and, recently,
cytoplasmic plasmid injection. The major impact of transgenic pigs
and minipigs took place in the fields of humanised pig models
and biomedical disease models, whereas agricultural applications
did not find broad acceptance. The recent release of the porcine
whole genome sequence and parallel developments of highly specific enzymes and RNAs now make it possible to perform precise
genetic modifications and fully exploit the advantages of this large
animal model. We anticipate that genetically modified pigs and
minipigs will increasingly complement the commonly used smallanimal models in biomedical research, since several aspects of
disease progression, physiology, metabolism and aging cannot
properly be mirrored in small-animal models.
Whitelaw, 2003; Niemann and Kues, 2007; Robl et al., 2007). A
bottleneck for porcine transgenesis is the lack of authentic pluripotent stem cells that are suitable for blastocyst complementation
experiments (Brevini et al., 2008; Kues et al., 2010a). The seminal development of induced pluripotent stem cells (iPS) in mice
and humans (Takahashi and Yamanaka, 2006) provides a new
approach to this end. The results of the first attempts to generate
porcine iPS cells were published recently (Esteban et al., 2009;
Wu et al., 2009; Ezashi et al., 2009), yet the potential of current
porcine iPS cells to contribute to chimera formation seems to be
limited (West et al., 2010).
This paper briefly discusses the current progress of transgenic
pig models for biomedical research. Comprehensive overviews
about transgenic pigs and livestock are available elsewhere (Clark
and Whitelaw, 2003; Robl et al., 2007; Kues and Niemann, 2011;
Whyte and Prather, 2011).
Introduction
Basic and biomedical applications
of transgenic pigs
The production of transgenic pigs is labour-intensive and costintensive and depends on advanced techniques in molecular biology and the micromanipulation of gametes and zygotes. At present,
progress in reproductive techniques and gene-transfer methods
has allowed targeted modifications of the porcine genome (glossary box), albeit the overall success rates are still low (Clark and
In the last few years, an expanded methodological repertoire for
porcine gene transfer has been developed (Table 1), resulting in an
increasing number of transgenic approaches (Whyte and Prather,
2011). At least 90% of genetically modified pigs are generated
for biomedical studies (Fig. 1A). Sequencing and annotation of
the porcine genome are important milestones for accelerating the
Fig.1. Increasing scientific interest in transgenic pig models
A) Scientific interest in porcine transgenesis. Depicted are the numbers of total citations per year, as extracted from
Thomson Reuters ISI Web of Knowledge for topic search terms “transgenic” and “pig model”. B) Transgenic boar exhibiting
ubiquitous expression of the Venus fluorophor gene (Garrels et al., 2011). The boar is shown under specific excitation conditions of Venus, in front of the boar an autofluorescent toy is visible. Almost all somatic and germ cells are fluorescent.
Key words: Domestic animals, disease model, humanised, genome, large animal model
Newsletter 36 Autumn 2011
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Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research
Table 1. Progress of technologies for transgenesis in pigs and minipigs
Development
Strategy
Reference
First transgenic pigs
PNI
Hammer et al., 1985
Somatic cloning of transgenic pigs
SCNT using transgenic donor cells
Park et al., 2001
Sperm-mediated gene transfer
SMGT
Lavitrano et al., 2002; Chang et al., 2002
Knock-out in pigs
Homologous recombination in somatic
cells and SCNT
Dai et al., 2002; Lai et al., 2002
Homozygous gene knockout
Homozygous knockout
Phelps et al., 2003
Lentiviral transgenesis
Perivitelline injection of lentiviruses
Hofmann et al., 2003; Whitelaw et al., 2004
SMGT / ICSI combination
SMGT and ICSI
Kurome et al, 2006
Conditional transgenesis
PNI
Kues et al., 2006
Episomal transgenesis
SMGT and episomal plasmid
Manzini et al., 2006; Giovannoni et al., 2010
Gene knock-down
Knock-down of PERV genes with siRNA and
SCNT
Dieckhoff et al., 2008; Ramsoondar et al.,
2009
Transposon transgenesis
Sleeping Beauty transposition in zygotic genome Garrels et al., 2010; Kues et al., 2010b
by CPI
Transposon transgenesis
Sleeping Beauty transposition in somatic cells
and SCNT
Jacobsen et al., 2011; Carlson et al. 2011
Targeted gene knockout
Zinc finger nuclease-catalysed gene deletion in
primary cells and SCNT
Whyte et al., 2011; Yang et al., 2011;
Hauschild et al., 2011
Targeted integration
Recombination-mediated cassette exchange in
primary cells and SCNT
Garrels et al., 2011
generation of transgenic models, even if the porcine genome
assembly still has gaps (annotated porcine genome data can be
found at: www.ensembl.org and www.pubmed.org). Since pig
and minipig physiology, anatomy, pathology, genome organisation, body weight and life span are more similar to humans than
are rodents, the domesticated pig represents a more appropriate
biomedical model (Table 2).
For certain biomedical therapies, such as xenotransplantation (transplantation of organs from one species to another (e.g.
porcine-to-human)), transgenic pigs are the only reasonable species (Niemann and Kues, 2003). Xenotransplantation seems to
be one option for closing the widening gap between demand and
availability of appropriate human organs (Yang and Sykes, 2007).
The prerequisites for potential porcine–human xenotransplantation are: (i) overcoming immunological hurdles; (ii) preventing the
transmission of porcine pathogens to human recipients; and (iii) the
compatibility of porcine organs with human physiology.
The suppression of hyperacute rejection of porcine xenografts
has been achieved by transgenic expression of human regulators of complement activity (RCA) (Tucker et al., 2002) and a
gene knockout of the porcine alpha, 1,3-galactosyltransferase
gene (Dai et al., 2002; Lai et al., 2002; Phelps et al., 2003).
Maximal survival rates of up to 3–6 months have been achieved
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Newsletter 36 Autumn 2011
with porcine alpha-galactosyltransferase knockout organs (kidney
or heart) transplanted to baboons (Kuwaki et al., 2005; Yamada
et al., 2005).
Extensive research has been conducted to reduce the risk of
porcine endogenous retrovirus (PERV) transmission to human
patients (Switzer et al., 2001; Irgang et al., 2003). RNA interference (RNAi) is a promising method for knocking down the PERV
expression. RNAi is based on small RNAs, either small interfering
RNA (siRNA) or short hairpin RNAs (shRNA). In the cytoplasm,
small RNA molecules are incorporated into an RNA-induced
silencing complex (RISC) and targets binding to a complementary
transcript sequence, resulting in mRNA degradation (Plasterk,
2002; Dallas and Vlassow, 2006). The efficacy of RNAi for reducing PERV expression has been demonstrated in cloned piglets
(Dieckhoff et al., 2008; Ramsoondar et al., 2009).
For several approaches, a conditional gene expression is desirable over a constitutive transgenic expression. Initial animal models carrying the first generation of conditional promoter elements
suffered from high basal-expression levels and pleiotropic effects
(Miller et al., 1989). Recent expression systems responsive to
exogenous tetracycline resulted in more tightly controlled expression. In pigs, a tetracycline-controlled transgenic expression was
achieved with a bicistronic expression cassette (Kues et al., 2006)
➤
that was designed to give ubiquitous expression of human RCAs.
Crossbreeding of lines with two cassettes was necessary to overcome epigenetic silencing and to achieve tetracycline-sensitive
RCA expression.
Transgenic pigs have been shown to mimic human diseases
such as atherosclerosis, non-insulin-dependent diabetes, cystic
fibrosis, cancer, ophthalmological and neurodegenerative disorders
(Kues and Niemann, 2004; Kragh et al., 2010; Rogers et al.,
2008; Yang et al., 2010; Luo et al., 2011). An important example is the minipig cystic fibrosis model, which develops disease
phenotypes that are highly similar to human patients (Rogers et
al., 2008), whereas transgenic mouse models failed to exhibit
lung, pancreatic and intestinal obstructions. Huntington’s disease
is a neurodegenerative disorder characterised by the expression
of mutated huntingtin with expanded polyglutamine tracts. The
misfolded protein accumulates in neurons and is suspected of triggering apoptosis. Whereas genetic mouse models often failed to
replicate overt neurodegeneration and apoptosis, a minipig model
expressing the N-terminal huntingtin with a polyglutamine tract
seems to do so (Yang et al., 2010).
Truncation mutations in the elongation of a very long-chain fattyacids-4 (ELOVL4) gene cause macular dystrophy. Photoreceptor
topography in the pig retina is more similar to that in humans as it
includes cone-rich, macula-like area centralis, whereas mice lack
a macular. Transgenic pigs expressing disease-causing ELOVL4
mutations were generated by PNI and SCNT (Sommer et al.,
2011). A detailed analysis showed photoreceptor loss, disorganised inner and outer segments, and diminished electroretinography responses, suggesting that the transgenic pigs mirror macular
degeneration and provide a unique model for therapeutic interven-
tion. Recently, the first immunodeficient pigs were cloned by SCNT
(Mendicino et al., 2010; Ramsoondar et al., 2011), promising to
serve as large-animal models for cell transplantation experiments.
Conventional gain-of-function transgenesis is based on random
integration of the transgene at sites of spontaneous double-strand
breaks of chromosomal DNA. The frequency of DNA doublestrand breaks at a defined locus can be considerably increased by
introducing specifically designed endonuclease enzymes (Urnov
et al., 2005; Arnould et al., 2007). The artificial endonucleases
are based on the DNA recognition sites of zinc finger transcription
factors, meganuclei or transcription factor like elements (TALE),
and they can be designed to bind highly specifically to a single,
predetermined sequence in the genome. Double-strand breakrepair pathways often create small deletions and, thus, designed
endonucleases allow efficient gene knockouts. The proof-of-principle to generate knockout pigs by synthetic zinc finger nucleases
has been demonstrated by the inactivation of enhanced green
fluorescent protein (EGFP), peroxisome proliferator-activated
receptor (PPAR gamma) and alpha-galactosyltransferase (Whyte
et al., 2011; Yang et al., 2011; Hauschild et al., 2011) in primary
somatic cells and the subsequent use of knockout cells for SCNT,
respectively. Thus current lack of authentic porcine ES cells can be
circumvented for the purpose of generating knockout pigs.
DNA-based transposons are mobile genetic elements that
move in the genome via a “cut-and-paste” mechanism. Most DNA
transposons are simply organised: they encode a transposase
protein flanked by inverted terminal repeats (ITRs), which carry
transposase binding sites, and it has been possible to separate
the transposase coding sequence from ITR sequences. Any DNA
flanked by ITRs will be recognised by the transposase and will
Table 2. Selected pig and minipig models for biomedicine and pharmaceutical research
Model
Comment
Reference
Xenotransplantation
knockout of alpha-galactosyltransferase
Lai et al., 2002; Dai et al., 2002
Xenotransplantation
expression of tumour necrosis factor ligand
Klose et al., 2005
Xenotransplantation
expression of human leukocyte antigen
Weiss et al., 2009
Xenotransplantation
PERV-knock down
Dieckhoff et al., 2008
Xenotransplantation
expression of human thrombomodulin
Petersen et al., 2009
Xenotransplantation
expression of human A20 (anti-apoptotic gene)
Oropeza et al., 2009
Cystic fibrosis pig
knockout of cystic fibrosis transmembrane conductance receptor
Rogers et al., 2008
Diabetes model
expression of mutated hepatocyte nuclear factor-1
Umeyama et al., 2009
Diabetes model
expression of mutated insulin 2
Renner et al., 2010
Immunodeficient pig
knockout of light chain
Ramsoondar et al., 2010
Immunodeficient pig
knockout of joining gene cluster
Mendicino et al., 2010
Huntington model
expression of mutated huntingtin with polyglutamine tract
Yang et al., 2010
Alzheimer model
expression of mutated human amyloid precursor protein
Kragh et al., 2010
Breast cancer
knockout of BRCA1 gene
Luo et al., 2011
Macular degeneration
introduced deletion in ELOVL4 gene
Sommer et al., 2011
Newsletter 36 Autumn 2011
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Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research
Fig.2. Applications of transposon transgenesis
Depicted is one integration site of a Venus transposon on chromosome X (red arrow). By means of targeted cassette exchange
(via the Cre/loxP system), the Venus reporter gene can be replaced by a gene of choice (I), thus introducing a transgene in a
pretested locus (Garrels et al., 2011) suitable for expression, and avoiding integration into heterochromatic regions or insertional mutagenesis. Alternatively, by supplying the SB transposase in trans, a remobilisation (II) of the transposon can be
induced. The annotated pig genome sequence was extracted from www.ensembl.org.
become enzymatically integrated into nuclear DNA. In a twocomponent system, the transposon is integrated solely by the
trans-supplementation activity of transposase. The first transposon
sufficiently active for use in vertebrates was the Sleeping Beauty
(SB) transposon (Ivics et al., 1997; Clark et al., 2007). Many
drawbacks of classical transgenic methods can be overcome
by transposition-catalysed gene delivery, which increases the
efficiency of chromosomal integration and facilitates single-copy
(monomeric) insertion events. An additional advantage of transposon-catalysed transgenesis is that the integration of monomeric
transgene units is directed to accessible euchromatic regions.
Transposon transgenic pigs have been generated (Kues et al.,
2010b; Garrels et al., 2011) by CPI (Iqbal et al., 2009), as well as
by SCNT (Jakobsen et al., 2010; Carlson, 2011; Garrels, 2011).
Ubiquitous expression of a fluorescent Venus protein, a derivative
of the commonly used EGFP, was found in somatic and germ
cells (differentiated spermatozoa) in own experiments (Fig. 1B,
Garrels et al., 2011) for all integrations sites, strongly supporting
the hypothesis that transposase preferentially integrates DNA into
euchromatic regions. The robust transgenic expression of Venus is
strictly copy-number dependent and facilitates cell-tracking experiments in cell-therapy approaches. The identification of integrations
sites revealed that most transposon integration sites were found in
intergenic regions of the porcine genome (Fig. 2). This approach
made it possible to identify loci, which are suitable for transgenic
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Newsletter 36 Autumn 2011
expression. Importantly, transposon-tagged loci can be readdressed by recombination-mediated cassette exchange (RMCE) in
cell culture. Via SCNT, the RMCE cells can be used to generate
vital piglets carrying a targeted integration into a “safe harbour”
locus (Garrels et al., 2011).
Since integrated transposons can be remobilised in the presence of a transposase enzyme, these animals can provide the
basis for performing whole genome mutagenesis screens in the
pig. For the SB transposon, the phenomenon of local hopping
after mobilisation has been described. The majority of secondary
integrations take place at a distance of up to 5 megabases from
the original integration. Figure 2 depicts one integration site on
the gene-rich X chromosome. The neighbouring porcine genes
are the von Hippel-Lindau binding gene (VBP1) and a novel gene,
both about 10,000 base pairs away from the integration site. After
mobilisation, the integration site can be screened for integration
events in neighbouring genes, such as the VBP1. The VBP1 gene
is of potential interest as an animal model, and the gene product is
assumed to form a complex with the von Hippel-Lindau tumor suppressor (VHL). The von Hippel-Lindau syndrome is a dominantly
inherited cancer syndrome predisposing carriers to several malignant and benign tumours. Thus, transposon transgenic pigs can be
employed for performing unbiased and biased mutagenic events.
It is anticipated that mutagenic screens with more advanced constructs will be applied in the near future.
➤
Conclusions
Methodological improvements for gene transfer into the pig
genome and a rapidly increasing list of biomedical pig models have
been developed in recent years. Together with more accurate
genome data and highly specific designed enzymes and RNAs,
precise genetic modifications have become feasible. It is anticipated that authentic pluripotent cells of the pig will be generated in
the near future. Thus, porcine transgenesis will become a routine
tool for generating relevant humanised porcine models. The most
obvious application of transgenic pigs will be as disease models
and biomedical therapies, which are not well-reflected in small
rodent models. The progress expected in porcine transgenesis
(increased success rates and decreasing costs), however, will
make the pig an attractive complementary model for advanced
approaches in biomedical research.
Acknowledgments
The expert technical support of Ms S. Holler, Ms Barg-Kues,
Ms Herrmann and Ms Ziegler, and the financial support of the
Deutsche Forschungsgemeinschaft (DFG) are gratefully acknowledged.
Conflicts of interest
The authors declare no conflicts of interest.
Wiebke Garrels, Heiner Niemann
Friedrich-Loeffler-Institute
Mariensee, DE-31535 Neustadt, Germany
Wilfried A. Kues
Friedrich-Loeffler-Institute
Institute of Farm Animal Genetics
Mariensee, DE-31535 Neustadt, Germany
0049 – (0)5034 871 120
0049 – (0)5034 871 101
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