Reprogramming Of Japanese Black Cattle Somatic Cells Incubated With Xenopus Laevis Oocyte Extract And Its Effects On The SCNT Efficiency

Somatic cell nuclear transfer (SCNT) has developed for nearly20years. However, to date, theeffciency of animal cloning is still extremely low. Relative studies have provided accumulatedevidences that the low efficiency of SCNT is due to the incomplete reprogramming and the aberrantepigenetic moclification of the highly differentiated donor cell nucleus. This study used Xenopus Leavisegg extract to induce japanese balck cattle fetal fibroblasts reprogramming to a low differentiated stateand aimed to evaluate cloning efficiency using treated cells as chromatin donors.Experiment1. Preparation of Xenopus oocyte extractIn this study, Xenopus Laevis were superovulated, the freshly laid eggs were collected andcentrifuged at10000rpm to acquire the mitotic Xenopus oocyte extract. The protein concentration ofthe extract was between25-35μg/μL for each experiment. SDS-PAGE showed the each extract had thesame composition. So we established a stable Xenopus oocyte extract system.Experiment2. Xenopus oocyte extract induced cells reprogrammingThe japanese balck cattle fetal fibroblast cell lines through tissue cultivation were eatablished inthis experiment. Growth curve model and chromosome karyotype analyzing showed the cell lines to bein exponential phase from3to6day and had the normal diploid karyotype. The fetal fibroblast celllines grew well.We used digitonin as a membrane permeabilization agent to treat fetal fibroblast cells. The optimalconcentration of digitonin was7μg/mL. PI dyeing showed the efficiency of membrane permeabilizationwas55%. The digitonin-permeabilized cells were incubated in extract for1hour, and subsequentlycultured in DMEM medium supplemented with Ca2+for membrane resealing. After24hours, byimmunofluorescence we finded that deacetylation of H3K9in extract treatment cells (ETCs) comparedwith the untreated cells. Cultured6-7days various cell aggregates began to form well-defned colonystructures. Alkaline phosphatase expressed in colonies. The result of Oct4immunofluorescence andwestern blot showed the colonies expressed Oct4factor. We then tested the pluripotency marker genesexpression in ETCs. Oct4and Nanog expression were detected in colonies by reversed transcript PCR,but not Sox2expression. For furture research, realtime quantitative PCR tested the expression level ofOct4, Nanog in ETCs at different times (4d、5d、6d), the result showed both gene had a promotion trendwith days increase.Experiment3. The production of SCNT embryos.We combined the micro-extrusion and zona-free fusion to produce SCNT embryos. We usedjapanese balck cattle fetal fibroblast cells cultured in DMEM medium as the donor cells to compare theefficiency of two different activation method (ionomycin+6-DMAP vs A23187+6-DMAP), the resultshowed there were no significant difference between two groups (cleavage rate; blastocyst rate,92.16%1.76vs92.28%2.14;23.21%1.63vs24.18%4.18). Then we used ETCs and untreated cellsas donor cells to compare the efficiency of SCNT, the result showed there were no significant betweentwo groups(fusion rate; cleavage rate; blastocyst rate,92.83%1.11vs96.04%1.52;89.64%±7.49vs 89.78±3.65;24.06%±6.91vs23.12%±6.80).