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AbstractThe first transgenic pigs were produced by the microinjection of foreign DNA into zygotic pronuclei in 1985. Since then, the me-thodological 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 spe-cific 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 small-animal models in biomedical research, since several aspects of disease progression, physiology, metabolism and aging cannot properly be mirrored in small-animal models.

introductionThe production of transgenic pigs is labour-intensive and cost-intensive and depends on advanced techniques in molecular biolo-gy 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 (glos-sary box), albeit the overall success rates are still low (Clark and

Whitelaw, 2003; Niemann and Kues, 2007; Robl et al., 2007). A bottleneck for porcine transgenesis is the lack of authentic pluri-potent stem cells that are suitable for blastocyst complementation experiments (Brevini et al., 2008; Kues et al., 2010a). The semi-nal 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).

basic and biomedical applications of transgenic pigsIn 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

Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical ResearchAuthors: Wiebke Garrels, Heiner Niemann. Corresponding author: Wilfried A. Kues

key words: Domestic animals, disease model, humanised, genome, large animal model

Fig.1. Increasing scientific interest in transgenic pig modelsA) 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 condi-tions of Venus, in front of the boar an autofluorescent toy is visible. Almost all somatic and germ cells are fluorescent.

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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 organisa-tion, 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 xenotransplanta-tion (transplantation of organs from one species to another (e.g. porcine-to-human)), transgenic pigs are the only reasonable spe-cies (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 xenotransplanta-tion 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 regula-tors 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

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 interfe-rence (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 redu-cing PERV expression has been demonstrated in cloned piglets (Dieckhoff et al., 2008; Ramsoondar et al., 2009).

For several approaches, a conditional gene expression is desi-rable over a constitutive transgenic expression. Initial animal mod-els 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 expres-sion. In pigs, a tetracycline-controlled transgenic expression was achieved with a bicistronic expression cassette (Kues et al., 2006)

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 by CPI

Garrels et al., 2010; Kues et al., 2010b

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

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that was designed to give ubiquitous expression of human RCAs. Crossbreeding of lines with two cassettes was necessary to over-come 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 exam-ple 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 trig-gering 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 fatty-acids-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, disorga-nised inner and outer segments, and diminished electroretinogra-phy 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 double-strand 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 break-repair pathways often create small deletions and, thus, designed endonucleases allow efficient gene knockouts. The proof-of-prin-ciple 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

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become enzymatically integrated into nuclear DNA. In a two-component 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 trans-poson-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 experi-ments 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

expression. Importantly, transposon-tagged loci can be read-dressed 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 pre-sence 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 sup-pressor (VHL). The von Hippel-Lindau syndrome is a dominantly inherited cancer syndrome predisposing carriers to several malig-nant 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 con-structs will be applied in the near future.

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 inser-tional 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.

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ConclusionsMethodological 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 anticipa-ted 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.

AcknowledgmentsThe 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 acknow-ledged.

Conflicts of interestThe authors declare no conflicts of interest.

Wiebke Garrels, Heiner NiemannFriedrich-Loeffler-InstituteMariensee, DE-31535 Neustadt, Germany

Wilfried A. KuesFriedrich-Loeffler-InstituteInstitute of Farm Animal GeneticsMariensee, DE-31535 Neustadt, Germany0049 – (0)5034 871 1200049 – (0)5034 871 101

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