Study On The Interspecies Somatic Nuclear Transfer Between Mouse And Rabbit

Based on the techniques of oocyte enucleation, activation and embryoreconstruction in mouse (or rabbit), the preimplantation development of interspeciesnuclear transfer embryos constructed from rabbit and mouse (or mouse and rabbit)were studied in this research in order to improve the early development of interspeciescloned embryos and set up the available interspecies somatic nuclear transfer methodsbetween mouse and rabbit.In the first part of this research, rabbit fetal fibroblast cell was used as donor andmouse enucleated oocyte was used as recipient oocyte. The results were as follow:1). The effects of several chemical activation procedures on the cleavage anddevelopment of mouse oocytes were investigated. The results indicated that mouseoocytes could be activated efficiently by 10mM Sr²⁺ for 4h, and oocytes at 20h afterHCG-injection were easily activated by 10mM Sr²⁺. Adding cytochalasin B inactivation medium was benefit to the activation and development of mouse oocytes.2). To obtain reliable stage donor cells, rabbit fetal fibroblast cells at passage 4-6were treated as follow respectively: cultured in 20μM Roscovitine for 24h; cultured in2μg/ml Aphidicolin for 24h, cultured in 2μM Nocodozole for 24h, cultured to fullconfluent state, serum starved for three days, serum starved for seven days. Theresults indicated that rabbit fetal fibroblasts at full confluent state had the highestpercentage of G₀+G₁, so that culture fibroblast cells to full confluence was the bestmethod to synchronize donors in G₀+G₁.3). Several different electrofusion parameters were tried to induce the fusionbetween rabbit fetal fibroblast and mouse enucleated oocyte to produce thereconstructed embryo. The results indicated the highest percentage of fusion could be obtained by two direct current pulses of 180V/mm for 10μsec each.4). Interspecies nuclear transfer embryos were reconstructed with mouseenucleated oocytes and rabbit fetal fibroblasts at full confluent state or at70%～80% confluent state. The results indicated the cleavage and blastocystdevelopment rates of reconstructed embryos derived from rabbit fetalfibroblast at full confluent state were higher than that of reconstructedembryos derived from 70%～80% confluent fibroblasts, though there was nosignificant difference between them (P＞0.05).5). The fact that interspecies nuclear transfer embryos reconstructed byrabbit fetal fibroblasts and mouse enucleated oocytes can develop toblastocysts in vitro demonstrated the mouse enucleated oocyte can supportrabbit somatic cell reprogramming and permit reconstructed embryos finishthe early development.In the second part of this research, mouse fetal fibroblastl was used as donorand rabbit enucleated oocyte was used as recipient oocyte. The results were as follow:1). To select the suitable culture condition of rabbit embryos, rabbitparthenogenetic embryos were cultured in Ham's F₁₂+BSA, 1640+BSA, M199+BSA,RD (1640:DEME=1:1)+BSA, RD+FBS, respectively. The results showed theblastocyst development rate of rabbit parthenogenetic embryos cultured in RD+FBSwas significantly higher than others. It indicated that RD+FBS was suitable for invitro culture of rabbit parthenogenetic embryos.2). Several different electrofusion parameters were tried to induce the fusionbetween mouse fetal fibroblast and rabbit enucleated oocyte to reconstruct theinterspecies nuclear transferred embryo. The results indicated the highest percentageof fusion between mouse fibroblasts and rabbit enucleated oocytes could be obtainedby two direct current pulses of 240V/mm for 20μsec each.3). The fact that interspecies nuclear transfer embryos reconstructed bymouse fetal fibroblasts and rabbit enucleated oocytes can develop toblastocysts in vitro demonstrated the rabbit enucleated oocyte can also supportmouse somatic cell reprogramming and make reconstructed embryos fulfill theearly development.