Production of transgenic blastocyst of sheep by somatic cell cloning  

作　　者：Xiaorong An Kemian Gou Yongfu Chen 

机构地区：[1]China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100094, Peoples R China

出　　处：《Chinese Science Bulletin》2001年第19期1630-1634,1674,共6页

基　　金：This work was supported by the National "863" Project (Grant No. Z21-01- 01).

摘　　要：Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had notshown difference significantly (P】0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experimentFive samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

关 键 词：SHEEP GRANULOSA cell NUCLEUS TRANSFER TRANSGENIC blastocyst. 

分 类 号：Q789[生物学—分子生物学]