Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

Authors: Wiebke Garrels, Heiner Niemann. Corresponding author: Wilfried A. Kues

Abstract Whitelaw, 2003; Niemann and Kues, 2007; Robl et al., 2007). A The first transgenic pigs were produced by the microinjection of bottleneck for porcine transgenesis is the lack of authentic pluri- foreign DNA into zygotic pronuclei in 1985. Since then, the me- potent stem cells that are suitable for blastocyst complementation thodological repertoire for porcine transgenesis was expanded to experiments (Brevini et al., 2008; Kues et al., 2010a). The semi- somatic cell nuclear transfer, lentiviral transgenesis and, recently, nal development of induced pluripotent stem cells (iPS) in mice cytoplasmic plasmid injection. The major impact of transgenic pigs and humans (Takahashi and Yamanaka, 2006) provides a new and minipigs took place in the fields of humanised pig models approach to this end. The results of the first attempts to generate and biomedical disease models, whereas agricultural applications porcine iPS cells were published recently (Esteban et al., 2009; did not find broad acceptance. The recent release of the porcine Wu et al., 2009; Ezashi et al., 2009), yet the potential of current whole genome sequence and parallel developments of highly spe- porcine iPS cells to contribute to chimera formation seems to be cific enzymes and RNAs now make it possible to perform precise limited (West et al., 2010). genetic modifications and fully exploit the advantages of this large This paper briefly discusses the current progress of transgenic animal model. We anticipate that genetically modified pigs and pig models for biomedical research. Comprehensive overviews minipigs will increasingly complement the commonly used small- about transgenic pigs and livestock are available elsewhere (Clark animal models in biomedical research, since several aspects of and Whitelaw, 2003; Robl et al., 2007; Kues and Niemann, 2011; disease progression, physiology, metabolism and aging cannot Whyte and Prather, 2011). properly be mirrored in small-animal models. Basic and biomedical applications Introduction of transgenic pigs The production of transgenic pigs is labour-intensive and cost- In the last few years, an expanded methodological repertoire for intensive and depends on advanced techniques in molecular biolo- porcine gene transfer has been developed (Table 1), resulting in an gy and the micromanipulation of gametes and zygotes. At present, increasing number of transgenic approaches (Whyte and Prather, progress in reproductive techniques and gene-transfer methods 2011). At least 90% of genetically modified pigs are generated has allowed targeted modifications of the porcine genome (glos- for biomedical studies (Fig. 1A). Sequencing and annotation of sary box), albeit the overall success rates are still low (Clark and 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 condi- tions 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 17 ➤ 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 Dai et al., 2002; Lai et al., 2002 cells and SCNT 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 Dieckhoff et al., 2008; Ramsoondar et al., SCNT 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 Jacobsen et al., 2011; Carlson et al. 2011 and SCNT Targeted gene knockout Zinc finger nuclease-catalysed gene deletion in Whyte et al., 2011; Yang et al., 2011; primary cells and SCNT Hauschild et al., 2011 Targeted integration Recombination-mediated cassette exchange in Garrels et al., 2011 primary cells and SCNT

generation of transgenic models, even if the porcine genome with porcine alpha-galactosyltransferase knockout organs (kidney assembly still has gaps (annotated porcine genome data can be or heart) transplanted to baboons (Kuwaki et al., 2005; Yamada found at: www.ensembl.org and www.pubmed.org). Since pig et al., 2005). and minipig physiology, anatomy, pathology, genome organisa- Extensive research has been conducted to reduce the risk of tion, body weight and life span are more similar to humans than porcine endogenous retrovirus (PERV) transmission to human are rodents, the domesticated pig represents a more appropriate patients (Switzer et al., 2001; Irgang et al., 2003). RNA interfe- biomedical model (Table 2). rence (RNAi) is a promising method for knocking down the PERV For certain biomedical therapies, such as xenotransplanta- expression. RNAi is based on small RNAs, either small interfering tion (transplantation of organs from one species to another (e.g. RNA (siRNA) or short hairpin RNAs (shRNA). In the cytoplasm, porcine-to-human)), transgenic pigs are the only reasonable spe- small RNA molecules are incorporated into an RNA-induced cies (Niemann and Kues, 2003). Xenotransplantation seems to silencing complex (RISC) and targets binding to a complementary be one option for closing the widening gap between demand and transcript sequence, resulting in mRNA degradation (Plasterk, availability of appropriate human organs (Yang and Sykes, 2007). 2002; Dallas and Vlassow, 2006). The efficacy of RNAi for redu- The prerequisites for potential porcine–human xenotransplanta- cing PERV expression has been demonstrated in cloned piglets tion are: (i) overcoming immunological hurdles; (ii) preventing the (Dieckhoff et al., 2008; Ramsoondar et al., 2009). transmission of porcine pathogens to human recipients; and (iii) the For several approaches, a conditional gene expression is desi- compatibility of porcine organs with human physiology. rable over a constitutive transgenic expression. Initial animal mod- The suppression of hyperacute rejection of porcine xenografts els carrying the first generation of conditional promoter elements has been achieved by transgenic expression of human regula- suffered from high basal-expression levels and pleiotropic effects tors of complement activity (RCA) (Tucker et al., 2002) and a (Miller et al., 1989). Recent expression systems responsive to gene knockout of the porcine alpha, 1,3-galactosyltransferase exogenous tetracycline resulted in more tightly controlled expres- gene (Dai et al., 2002; Lai et al., 2002; Phelps et al., 2003). sion. In pigs, a tetracycline-controlled transgenic expression was Maximal survival rates of up to 3–6 months have been achieved achieved with a bicistronic expression cassette (Kues et al., 2006)

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that was designed to give ubiquitous expression of human RCAs. tion. Recently, the first immunodeficient pigs were cloned by SCNT Crossbreeding of lines with two cassettes was necessary to over- (Mendicino et al., 2010; Ramsoondar et al., 2011), promising to come epigenetic silencing and to achieve tetracycline-sensitive serve as large-animal models for cell transplantation experiments. RCA expression. Conventional gain-of-function transgenesis is based on random Transgenic pigs have been shown to mimic human diseases integration of the transgene at sites of spontaneous double-strand such as atherosclerosis, non-insulin-dependent diabetes, cystic breaks of chromosomal DNA. The frequency of DNA double- fibrosis, cancer, ophthalmological and neurodegenerative disorders strand breaks at a defined locus can be considerably increased by (Kues and Niemann, 2004; Kragh et al., 2010; Rogers et al., introducing specifically designed endonuclease enzymes (Urnov 2008; Yang et al., 2010; Luo et al., 2011). An important exam- et al., 2005; Arnould et al., 2007). The artificial endonucleases ple is the minipig cystic fibrosis model, which develops disease are based on the DNA recognition sites of zinc finger transcription phenotypes that are highly similar to human patients (Rogers et factors, meganuclei or transcription factor like elements (TALE), al., 2008), whereas transgenic mouse models failed to exhibit and they can be designed to bind highly specifically to a single, lung, pancreatic and intestinal obstructions. Huntington’s disease predetermined sequence in the genome. Double-strand break- is a neurodegenerative disorder characterised by the expression repair pathways often create small deletions and, thus, designed of mutated huntingtin with expanded polyglutamine tracts. The endonucleases allow efficient gene knockouts. The proof-of-prin- misfolded protein accumulates in neurons and is suspected of trig- ciple to generate knockout pigs by synthetic zinc finger nucleases gering apoptosis. Whereas genetic mouse models often failed to has been demonstrated by the inactivation of enhanced green replicate overt neurodegeneration and apoptosis, a minipig model fluorescent protein (EGFP), peroxisome proliferator-activated expressing the N-terminal huntingtin with a polyglutamine tract receptor (PPAR gamma) and alpha-galactosyltransferase (Whyte seems to do so (Yang et al., 2010). et al., 2011; Yang et al., 2011; Hauschild et al., 2011) in primary Truncation mutations in the elongation of a very long-chain fatty- somatic cells and the subsequent use of knockout cells for SCNT, acids-4 (ELOVL4) gene cause macular dystrophy. Photoreceptor respectively. Thus current lack of authentic porcine ES cells can be topography in the pig retina is more similar to that in humans as it circumvented for the purpose of generating knockout pigs. includes cone-rich, macula-like area centralis, whereas mice lack DNA-based transposons are mobile genetic elements that a macular. Transgenic pigs expressing disease-causing ELOVL4 move in the genome via a “cut-and-paste” mechanism. Most DNA mutations were generated by PNI and SCNT (Sommer et al., transposons are simply organised: they encode a transposase 2011). A detailed analysis showed photoreceptor loss, disorga- protein flanked by inverted terminal repeats (ITRs), which carry nised inner and outer segments, and diminished electroretinogra- transposase binding sites, and it has been possible to separate phy responses, suggesting that the transgenic pigs mirror macular the transposase coding sequence from ITR sequences. Any DNA degeneration and provide a unique model for therapeutic interven- 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 19 ➤ 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 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.

become enzymatically integrated into nuclear DNA. In a two- expression. Importantly, transposon-tagged loci can be read- component system, the transposon is integrated solely by the dressed by recombination-mediated cassette exchange (RMCE) in trans-supplementation activity of transposase. The first transposon cell culture. Via SCNT, the RMCE cells can be used to generate sufficiently active for use in vertebrates was the Sleeping Beauty vital piglets carrying a targeted integration into a “safe harbour” (SB) transposon (Ivics et al., 1997; Clark et al., 2007). Many locus (Garrels et al., 2011). drawbacks of classical transgenic methods can be overcome Since integrated transposons can be remobilised in the pre- by transposition-catalysed gene delivery, which increases the sence of a transposase enzyme, these animals can provide the efficiency of chromosomal integration and facilitates single-copy basis for performing whole genome mutagenesis screens in the (monomeric) insertion events. An additional advantage of trans- pig. For the SB transposon, the phenomenon of local hopping poson-catalysed transgenesis is that the integration of monomeric after mobilisation has been described. The majority of secondary transgene units is directed to accessible euchromatic regions. integrations take place at a distance of up to 5 megabases from Transposon transgenic pigs have been generated (Kues et al., the original integration. Figure 2 depicts one integration site on 2010b; Garrels et al., 2011) by CPI (Iqbal et al., 2009), as well as the gene-rich X chromosome. The neighbouring porcine genes by SCNT (Jakobsen et al., 2010; Carlson, 2011; Garrels, 2011). are the von Hippel-Lindau binding gene (VBP1) and a novel gene, Ubiquitous expression of a fluorescent Venus protein, a derivative both about 10,000 base pairs away from the integration site. After of the commonly used EGFP, was found in somatic and germ mobilisation, the integration site can be screened for integration cells (differentiated spermatozoa) in own experiments (Fig. 1B, events in neighbouring genes, such as the VBP1. The VBP1 gene Garrels et al., 2011) for all integrations sites, strongly supporting is of potential interest as an animal model, and the gene product is the hypothesis that transposase preferentially integrates DNA into assumed to form a complex with the von Hippel-Lindau tumor sup- euchromatic regions. The robust transgenic expression of Venus is pressor (VHL). The von Hippel-Lindau syndrome is a dominantly strictly copy-number dependent and facilitates cell-tracking experi- inherited cancer syndrome predisposing carriers to several malig- ments in cell-therapy approaches. The identification of integrations nant and benign tumours. Thus, transposon transgenic pigs can be sites revealed that most transposon integration sites were found in employed for performing unbiased and biased mutagenic events. intergenic regions of the porcine genome (Fig. 2). This approach It is anticipated that mutagenic screens with more advanced con- made it possible to identify loci, which are suitable for transgenic structs will be applied in the near future.

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Conclusions transposon system. Transgenic Res. 20, Jan. 9 (epub ahead of Methodological improvements for gene transfer into the pig print). genome and a rapidly increasing list of biomedical pig models have Clark J, Whitelaw B (2003) A future for transgenic livestock. Nat been developed in recent years. Together with more accurate Rev Genet 4:825-833 genome data and highly specific designed enzymes and RNAs, Clark, K.J:, Carlson, D.F., Fahrenkrug, S.C. 2007. Pigs taking precise genetic modifications have become feasible. It is anticipa- wings with transposons and recombinases. Genome Biol. 8, Suppl ted that authentic pluripotent cells of the pig will be generated in 1: S13. the near future. Thus, porcine transgenesis will become a routine Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, tool for generating relevant humanised porcine models. The most Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell- obvious application of transgenic pigs will be as disease models Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL (2002) and biomedical therapies, which are not well-reflected in small Targeted disruption of the a1,3-galactosyltransferase gene in rodent models. The progress expected in porcine transgenesis cloned pigs. Nat Biotechnol 20: 251-255 (increased success rates and decreasing costs), however, will Dallas A, Vlassow A (2006) RNAi: A novel antisense technology make the pig an attractive complementary model for advanced and its therapeutic potential. Med Sci Monit 12 RA67-74 approaches in biomedical research. Dieckhoff B, Petersen B, Kues WA, Kurth R, Niemann H, Denner J (2008) Knockdown of porcine endogenous retrovirus Acknowledgments (PERV) expression by PERV-specific shRNA in transgenic pigs. The expert technical support of Ms S. Holler, Ms Barg-Kues, Xenotransplantation 15: 36-45 Ms Herrmann and Ms Ziegler, and the financial support of the Esteban, M.A., J. Xu, J. Yang, M. Peng, D. Qin, W. Li, Z. Jiang, Deutsche Forschungsgemeinschaft (DFG) are gratefully acknow- J. Chen, K. Deng, M. Zhong, J. Cai, L. Lai, and D. Pei. 2009. ledged. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem. 284:17634-17640. Conflicts of interest Ezashi, T., B.P.V.L. Telugu, A.P. Alexenko, S. Sachdev, S. The authors declare no conflicts of interest. Sinha, and R.M. Roberts. 2009. Derivation of induced pluri- potent stem cells from pig somatic cells. Proceedings of the Wiebke Garrels, Heiner Niemann National Academy of Sciences of the United States of America. Friedrich-Loeffler-Institute 106:10993-10998. Mariensee, DE-31535 Neustadt, Germany Garrels, W., Mates, L., Holler,S., Niemann, H., Izsvak, Z., Ivics, Z., Kues, W.A: 2010. Generation of transgenic pigs by the Wilfried A. 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