Collaboration: Maria SIBILIA, Anton WUTZ, Josef PENNINGER
Fusion of blastomeres of 2-cell embryos results in the generation of embryos with a tetraploid genome content. These embryos can develop to the blastocyst stage. However, tetraploid cells are excluded from the embryo proper. Using tetraploid morula stage embryos aggregated with ES cells led to the first demonstration that ES cell have the ability to form all embryonic structures (Nagy & Rossant). The entire mouse embryo is derived from ES cells, while the extraembryonic structures are formed from the tetraploid cells. Injection of wild type early passage ES cells into tetraploid blastocysts has subsequently been demonstrated to result in life born mice (Wang et al.). Our group has now established XY male and XX female F1 (B6x129J) mouse ES cell lines that are robust enough to generate healthy live offspring at high frequency even after injection into tetraploid blastocysts. We plan to generate other F1 mouse strain combinations for the generation of tetraploids and attempt to establish also robust inbred ES lines to avoid backcrossing. Since we currently perform injection of ES cells and use time consuming techniques to generate tetraploids, we will test and implement automated electrofusion and high-throughput aggregation technologies. Once established, we plan to switch all our targeting systems to tetraploids and generate novel and unique tools such as ES libraries for switchable and inducible transgenes (see below projects headed by M. Sabilia and A. Wutz) that will be essential for high through-put in vivo RNAi gene knockdown experiments (group Penninger). It should be noted that we will make all our tetraploid ES cell lines and reagents available to the research community.
