The Research On Transferred Cherry Gene Tan Sheep Somatic Cell Nuclear Transfer

Tan sheep are mutton-wool sheep as a features variety in Ningxia, However, the germplasm resources protection work has been delayed. The Tan sheep lambing rate is low and the number of purebred Tan sheep is dowm in recent years in Ningxia. In this study, the Tan ovarian collected from slaughterhouse, following the Tan sheep oocytes maturation in vitro, and the parthenogenetic embryos and reconstructed embryos were cultured in vitro. The Tan sheep oocytes was cultured in medium added with different concentration of HMG, FSH, LH. The oocyte was activated by the chemistry way of adding with ionomycin (Ionomycin) and unite6-diamino-purine (6-DMAP), and the phsics way of acting with different electrical activation parameters, and the Tan sheep parthenogenetic embryonic and transferred Cherry genes fibroblasts-oocytes reconstructed embryos was cultured in vitro. Also, the fusion rate was analyzed in different fusion conditions, and the embryo of cleavage rate, morula and blastocyst rate was done with statistical analysis. These results lay a foundation for the further study of the Ningxia Tan sheep cloning, which will be promoting the Tan sheep breeding and germplasm resources protection work in Ningxia. After experimenting, the following results was shown in this paper:1The Tan sheep fetal fibroblast cell line was established successfully by the tissue cultivation, and3male foetus cell lines of Tan sheep identified with SRY gene were acquired. The Cherry gene transfected in Tan sheep fibroblasts was stably expressed. And the karyotype of Tan sheep fetal fibroblast cells was analyzed in5th,15th and25th passage, and no chromosome deletion.2. The number of9138Tan sheep ovary was collected from slaughterhouse and the27607immature oocytes were cultured. The result showed that the mature rate was no significant difference in basical medium (adding10mg/mL BSA,100X ITS,1.5μg/mL17β-E2,2.5mmol/mLglutamine,1mmol/L sodium pyruvate in TCM199medium) adding with0.1IU/mL HMG or200μg/mL FSH and40μg/mL LH, which were69.6%and68.8%differently.3. Tan sheep oocytes activated by the both way of chemical and electrical. The cleavage rate and blastocyst development rate were81.0%and26.2%differently in6-DMAP and ionomycin, and which were58.5%and19.5%with1600V/cm, and74.1%and19.6%with6-DMAP and1600V/cm. The development rate of Tan sheep blastocyst was25.0%when cultured in mSOFaa medium, and which was singnificantly higher than16.7%in M16mediumm, or15.8%in KSOM medium. These results were showed that the ideal activation method is by the chemical way of6-DMAP and ionomycin, and mSOFaa media is suitable for Tan sheep embryos culture in vitro.4. The different parameters between two electrode in electric fusion has different effect on the development of Tan sheep oocytes. The electric fusion was1400V/cm-2000V/cm. The appropriate electric fusion parameter for Tan sheep somatic cell nuclear transfer embryo is1800v/cm of pulse intensity, the fusion rate is58.3%, the cleavage rate is40.0%, and the blastocyst rate is21.4%. and tow Cherry embryos were acquired in440reconstructed embryos.