Document Type : Original Article
Stem Cell Dept., Royan Institute, Tehran, Iran
Introduction: Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene functions in vivo and differentiation into specific types as potential therapeutic agents for several diseases. We describe here production of transgenic chimeric mouse blastocysts by aggregation of tetraploid embryos and embryonic stem cells expressing histone variant (H2A.Z) fused with EGFP.
Materials and Methods: To optimize electrofusion, two-cell mouse (NMRI) embryos were fused together by different voltages and durations. In optimumstate, tetraploid embryos were aggregated by genetically manipulated mouse embryonic stem cells (Royan B1) derived from C57BL/6. Electroporation used for insertion of vector included H2A.Z-EGFP. The aggregated embryos were cultured to blastocycts in vitro.
Results: Mouse blastomere fusion and tetraploid blastocyst development occurred by 100v and 25µs. 82% of tetraploid embryos reached blastocyst and hatching blastocyst stages. Also 75% of embryos aggregated together and 42% of them expressed H2A.Z-EGFP.
Conclusion: It is possible to use genetic manipulated ES cells and tetraploid embryos to study gene function.