The Culture Determinnation And The Effects On Nuclear Transfer Of Goat Fetal Fibroblast Cells

Experment 1:In this present study, we used SRY-PCR in cell gender identification, and investigate effects of cell cycle inhibitors: roscovitine and DMSO on the goat fetal fibroblast cells. The result of SRY-PCR indentified 3 female cell lines ,and 1 male lines. After the treatment of15uM roscovitine for 48 h, the percentage of fibroblast cells arrested in G0/G1 phases was 87.70%, but the percentage of cells arrested in G0 phases is not significantly increased (6.84%). 1% DMSO for 48h could arrests 32.54% fibroblasts in G0 phases. The results of TUNEL assay show that compared with the control group(1.12%), cell cycle inhibitors did not induced higher rates of cell apoptosis(roscovitine 1.42% , DMSO 0.9%).These results indicated that the SRY-PCR could be used in cell gender identification, and the use of cell cycle inhibitors may be a more effective and healthy in synchronization of donor cells.Experment 2: The present study was to investigate the differences of donor cell lines derived from the same tissue but different animal sources to support development of cloned embryos , and the differences in the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos. Four cell lines isolated respectively from four goat fetuses were used as donor cells in SCNT. The development of SCNT embryos show a significant difference among the four groups in blastocyst rates(16.46±0.42%, 16.13±1.26%, 17.02±1.54% VS 7.69±0.28%.) . This four fetal fibroblasts are transfected with PEGFP - C1, positive cell clones and amplified positive cell clones are compared after G418 screening. The results show that the number of positive clones and amplified cell clones of GFF1 and GFF2 are significantly higher than GFF3 and GFF4 (P < 0.05). The two strains chosen from GFF1 clones are used as donor cells, and compared by GFF1, there is no significant difference in blastocyst rates among three groups. The results show that different sources of the fetal fibroblasts also effect the development of cloned embryos ,and the efficiency of transfected exogenous gene on the developmental competence of goat SCNT embryos.Experment 3: The present study was to investigate effects of sequential media G1/G2 and modified synthetic oviductal fluid (mSOF) culture systems on developmental competence, cell apoptosis and the pregnancy of goat somatic cell nuclear transfer (SCNT) embryos. Goat NT embryos were cultured as following: (1) 72 h in G1 then 120 h in G2.3 (group G1G2), both the culture media supplemented with 0.8% BSA (bovine serum albumin); (2) 72 h in mSOF supplemented with 0.8 % BSA then in mSOF supplemented with 10% FBS (fetal bovine serum) for 120 h (group mSOF-FBS). The results indicated that there was no significant difference between the sequential media G1/G2 and mSOF-FBS in blastocyst rates (27.7±3.1% vs 25.3±1.0%,P>0.05). Both numbers of total cell and apoptotic nuclei in blastocyst from G1/G2 were significantly higher than mSOF-FBS (109.1±6.2 vs 93.2±4.5 and 11.3±0.1% vs 4.9±0.2% respectively, P<0.05 ). However, the pregnant rates on Day 30 in group G1/G2 were higher than those in group mSOF-FBS(21.4% vs 8.0%, P<0.05 ). In conclusion, compared to mSOF-FBS, the sequential media G1/G2 could better support the development of goat somatic cell nuclear transfer embryos.