Advanced transgenic strategies for modification of donor pigs in xenotransplantation

Authors


Abstract

For xenotransplantation diverse rejection mechanisms are much more pronounced as compared to allotransplantation. The usage of genetically modified pigs, however, facilitates tailoring of donor animals for defined purposes (1). Although such genetic modifications were done decades ago, routine generation of transgenic pigs was not performed until somatic cell nuclear transfer (SCNT) was implemented for reproduction of large animals. SCNT avoids the production of mosaic founders and shifted the genetic modification towards the level of pig primary cells and, thus, improved the efficiency of transgenesis as it enables the generation of almost exclusively transgenic offspring, once the donor cells have been properly selected for vector integration (2).

In addition, SCNT opened up the possibilities for any type of genetic modifications that has been developed for embryonic stem cells. In particular, the opportunity for site-directed mutagenesis boosted the potential of genetically modified pig models. This was demonstrated for the removal of the α1,3-galactosyl-galactose epitopes by disruption of the GGTA1 gene which reduced the problem of hyperacute rejection to a minor topic in the xenotransplantation community. In the meanwhile more sophisticated methods such as modified bacterial artificial chromosomes, viral vectors or site-specific nucleases further increased the potential for site-directed mutagenesis in pig (3, 4). The latter technology is based on the introduction of a DNA double strand break by a nuclease that is directed to the target site by specific DNA-binding domains. Mutations are introduced by erroneous repair through non-homologous end joining. Alternatively, a targeting vector can be used in combination with a site-specific nuclease to introduce a targeted modification via homologous recombination. Other advanced transgenic strategies such as the two-vector based TetOn technology for inducible transgene expression are routinely performed in the mouse, but the significantly longer generation time of large animals hampered its straight translation into the pig. As we demonstrated recently, sequential transgenesis by repeated rounds of SCNT is a practicable way to evaluate biological transgene function in founder animals within a considerable time frame (5).

In addition to technological improvements at the cellular as well as at the embryonic level, the recent boost of genomic information from multiple species and its bioinformatics analysis improved the design of transgenic pigs. As for many problems cell type-specific expression of a transgene is desired, the definition of appropriate regulatory elements is required. Many of those have been described in the mouse, but in general endogenous sequences are seen as superior to the usage of murine promoters in the pig. Multiple-sequence alignments from diverse mammalian species facilitate the identification of the orthologous region of murine regulatory elements in the pig.

Interestingly, with the increasing number of transgenes available for xenotransplantation approaches, the breeding aspect gained new attention. It is clear that for optimized donor pigs multiple transgenes should be combined and, on the long run, mendelian transgene segregation should be avoided by using novel transgene approaches. However, until such “all-in-one” vectors are available, the most straightforward strategy is the combination of existing and properly characterized lines by conventional breeding strategies. These require profound organization and logistics to resolve the conflicting aspects of transgene segregation and inbreeding and to enable the systematic evaluation of donor herds for microbial contamination.

Thus, the task field of donor pig suppliers in xenotransplantation expanded from relatively simple reproductive stints to advanced design and construction of novel transgenic pigs and organizing challenges regarding continuous supply of donor animals.

References 1. Klymiuk N, Aigner B, Brem, et al. Genetic modification of pigs as organ donors for xenotransplantation. Mol Reprod Dev 2010; 77: 209.

2. Aigner Bet al. Transgenic pigs as models for translational biomedical research. J Mol Med (Berl) 2010; 88, 653.

3. Klymiuk Net al. Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl) 2011.

4. Hauschild Jet al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proceedings of the National Academy of Sciences of the United States of America 2011; 108: 12013.

5. Klymiuk Net al. First inducible transgene expression in porcine large animal models. FASEB J 2011.

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