Study On Transgenic Cashmere Goat By Somatic Cell Nuclear Transfer

It is just beganning of somatic cell nuclear transfer (SCNT) on cashmere goat, here we established the SCNT on transgenic cashmere goat, and the influence of cell types on cloning efficiency of cashmere goat was investigated. Finally the identification and production performance testing of cloned transgenic cashmere goats were proceeded.1The establishment of cashmere goats SCNT:The fibroblast cells from four caprine fetals(caprine fetal fibroblast cells,CFC) and2adults ears(caprine ear fibroblast cells, CEFC) were cultured, and the genders of them were distinguished by SRY-PCR. Transgenic caprine fetal fibroblast cells(transgenic caprine fetal fibroblast cells, TCFC) were obtained after transfected with eukaryotic expression vector and screened with G418. Red fluorescence could be detectable in the TCFCs, and the results of PCR demonstrated the exogous gene was integrated stably. SCNT was performed with these TCFCs utilized as the nucleus donors. The rate of oocyte IVM was72.6%(420/578), and419reconstructed embryos were harvested. In the piriod of in vitro development,86.1%(361/419) oocyte cleavage rate and14.8%(62/419) blastula rate were obtained, while the red fluorescence could be obseved in all blastulas. Another SCNT and embryo transfer were performed in2009. Here we harvested6732oocytes,65.1%(4380/6732) of them had matured in vitro,and3568enucleated oocytes were fusioned with TCFCs and CFCs, the rate of fusion and cleavage were62.8%(2241/3568) and54.9%(1231/2241) respectively. Totally924embryos were transfered into273recipients, and20kids were born.2.The affect of donor cell types and recipients condition on cashmere goat SCNT efficiency:The efficiency of donor cell types in cashmere goat SCNT was undefined. In this study, SCNT was performed using CFCs, CEFCs and TCFCs as donor cells to compare the influences of cell type, transgene and sex of nuclear donor fibroblast cells on the SCNT efficiency including fusion and cleavage rates of reconstructed embryos and the birth, postnatal survival and mortality rates of cloned kids. A total of4943reconstructed embryos were obtained. Among them,3949embryos were fused, and3737embryos were cultured in vitro leading to a total of3094cleavage embryos. Furthermore, embryo transplantation analysis was conducted on1873cloned embryos with relatively normal morphology, and368recipient goats were transplanted and48kids were born, of which,35kids survived. The SCNT efficiency of CFCs cell-derived embryos was higher than that of CEFCs-derived embryos, and the cloning efficiency of non-transgenic cloned embryo was higher than that of transgenic cloned embryos. Also, the SCNT efficiency of male cell-derived embryos was higher than that of female cell-derived embryos. High oocytes cleavage rates predicted high birth rates and postnatal survival rates. The results indicated that the donor cell types of cashmere goats affected the clone efficience, and connections existed between the clone embryos early development ability and the SCNT efficience. The physiology condition (ovulation number) of recipients also affected the clone efficience.3. The Identification and production performance testing of cloned transgenic cashmere goats:The days of gestation and weight of newborn of cloned kids from different cells were recorded and analyzed. We also identified the transgenic kids and tested the production preformance. There was difference between the kids from various cells on days of gestation:the ones cloned from RCFC4was the longest,157.5days, while the ones cloned from ordinary somatic cells was the shortest,152.3days. Even though the kids cloned with same donors were various in days of gestation and weight of newborn, and this was more obvious in ones from transgenic cells. The red fluorescence could be observed on phenotype of some kids and the cells separated from ear apex tissues of these ones, while it could be detected in cells rather than phenotype of some others, and no fluorescence could be found in both cells or phenotype of remains, although the the IC and Neo fragment included in the exogous gene could be amplified from all cells. In the production performance testing of goats cloned from ordinary, transfected with IGF-1and VEGF CFC3cells, we found that the VEGF transfected goats had the greater improvement on cashmere production.