Derivation of livestock stem cells for applications in basic research, functional genomics, reproductive technology, biotechnology and biomedicine
Final Report Abstract
Our group focuses on generating genetically modified large animals for biomedicine, primarily pigs and rabbits. This requires a technology platform for the genetic manipulation of cultured cells, particularly gene targeting, the isolation, expansion and analysis of cell clones and the production of viable fertile animals. In mice, gene targeting in embryonic stem (ES) cells is now routine and many hundreds of gene-targeted animals have been produced. The situation is very different for pigs and rabbits. At the time of this application only one gene (a1,3 galactosyltransferase) had been targeted in pigs. The recent report of CFTR targeting has increased this to two. There has been no report of gene targeting in rabbits. This is not due to lack of interest in these species, on the contrary, but to difficulties such as the lack of ES cells, low gene targeting efficiency in somatic cells, poor proliferation of single cell clones and inefficient nuclear transfer, particularly in rabbits. This project sought to address these problems. We attempted derivation of rabbit and porcine pluripotent stem cells, applying recent knowledge gained from human stem cell culture and newly discovered exogenous and endogenous factors. Several putative rabbit ES cell lines were isolated, one of which produced a chimeric animal. In pig we investigated ES and induced pluripotent stem (iPS) cells. Porcine early passage ES cells could be isolated, but not maintained undifferentiated for long periods. We isolated porcine cell clones with iPS cell characteristics closely resembling iPS-like porcine cells described by other groups. Our results accord with recent publications in this field, in that these cells remain dependent on expression of exogenous transgenes to remain undifferentiated. We have isolated multipotent mesenchymal stem cell (MSCs) from rabbit and pig. These cells proliferated for long periods in culture, genetically manipulated cell clones could be derived and transgenic pigs and rabbits obtained by nuclear transfer. To our knowledge this is the second report of production of cloned transgenic rabbits. Gene-targeting experiments will soon proceed in the rabbit and gene targeted pigs derived from porcine MSCs have been produced. Gene targeting efficiencies up to 75% were obtained in pigs, depending on the cell isolate. Some targeted MSC clones retain their multipotent phenotype in vitro. The technology we have developed may have wider application. MSCs are already used for human cell transplantation, efficient gene targeting methods could therefore be important for human gene therapy.
Publications
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(2009) Nuclear transfer with rabbit embryonic stem cells: serum-starvation improves the quality of cloned embryos. Reproduction Fertility and Development 21: 129
Zakhartchenko V, Flisikovska F, Hao R, Li S, Kind A, Wolf E and Schnieke A
