The Influences Of Passage Times And BFGF On Ovine Embryonic Stem-like Cells And Production Of Mouse Tetraploid Blastulas

The reported livestock ESC-like are unable to achieve long-term culture, which restrict the research in livestock ESC. By detecting the oESC-like which established in our lab, we get the preliminary reasons that the oESC-like can not survive in long-term culture, lay the foundation for future research. In this sdudy, we observe the morphological characteristics of oESC-like, detect the expression of AKP, detect the expressions of PCNA、Bcl-2、Bax、Sox-2、Oct-4on protein level with iImmunofluorescence technic, and count the protein expressions with softwear named Image-Pro Plus6.0. The results showed that, the oESC-like kept the morphological characteristics of ESC in long-term culture, alkaline phosphatase expressed in every passages; the expression of Sox-2and Oct-4increased; the ability of proliferation had no significant change; the expression of Bax increased passage by passage, the expression of Bcl-2increased on passage10, and could inhibited the activation of Bax, then decreased significantly on passage15and20, and could not inhibited the activation of Bax; the ratio of Bcl-2/Bax decreased passage by passage, indicated that, the ovine embryonic stem-like cells were apoptosising gradually in long-term culture. The significance of this study is to provide a theoretical basis for the genetics and breeding, genetic engineering, medical, and other in practice.bFGF could promote the proliferation of ESC, maintain the undifferenct status of oESC-like. by increasing the concentration of bFGF, expression level of PCNA(P<0.05) was increased, the proliferation of oESC-like was promoted. Expression level of Bax was increased in passage5,10,15and20in higher concentration of bFGF culture system. Expression level of Bcl-2in higher concentration of bFGF culture system was same in passage5wiht the lower concentration of bFGF culture system(P>0.05), but increased in passage10,15and20compared with the lower concentration of bFGF culture system(P<0.05). The ratio of Bcl-2/Bax was stable in long-term culture. AKP was expressed strongly in higher concentration of bFGF culture system, expression level of Oct-4and Sox-2significantly increased in higher concentration of bFGF culture system(P<0.05).Tetraploid embryos have the most importance in mammalian gametogenesis, development, generation of tetraploid, heredity, immunity mechanism, so it is crucial to increase the rates of electrofusion and development. For searching the best method of electrofusion of mouse2-cell embryos, we used two different electrofusion solution(0.3M Mannitol solution and0.3M Sucrose solution) and three different electrofusion parameter(120V/mm,20μs×2、100V/mm,40μs×2、80V/mm,80μs×2)in each electrofusion solution. In the study, we got the highest fusion rate and blastocyst rate(92.5%and90.6%), and the best electrofusion solution is0.3M Mannitol and the best electrofusion parameter is80V/mm,80μs×2.