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Culture of bovine fetal Skin fibroblasts in vitro and Ipr1 Gene Transfection

畜牧兽医学报 2011年第S1期 7-12页

作者： SONG Yong-li 1,2,HE Xiao-ning 2,HUA Song 2,LAN Jie 2,ZHENG Yue-mao 2,LI Ji-xia 2,HE Xiao-ying 2,QUAN Fu-sheng 2,ZHANG Yong 2(1. College of Veterinary Medicine,Jilin Agricultural Science and Technology College,Jilin132101,China 2. Key Laboratory of Animal Reproductive Physiology & Embryo Technology,Ministry of Agriculture,College of Veterinary Medicine,Northwest Agricuture and Forestry University,Yangling 712100,China)

In order to generate transgenic donor cells for nuclear transfer, bovine fetal fibroblasts were isolated in vitro and transfected with the eukaryotic expression vector pSRA-EGFP-Ipr1. The mouse Ipr1 gene and human SR-A promoter were successfully cloned and then used to construct this macrophage-specific eukaryotic expression vector. bovine fetal fibroblasts in stable primary culture (4th passage) were transfected with pSRA-EGFP-Ipr1 by electroporation. Fluorescence from GFP was observed after 24h. Transgenic cells were selected using G418 and the resultant monoclones were picked and expanded. The transgenic cells, at the 9 th passage, were evaluated by PCR and flow cytometry. The inserted Ipr1 was confirmed by PCR, indicating stable integration of the transgene into the genome and cells had normal karyotypes and very good appearance, which indicate no deleterious result of the transgenesis. In conclusion, the cells obtained could be used as donor cells for nuclear transfer for further research of transgenic cattle.

关键词： donor cells eunuclear expression vector Ipr1 gene bovine fetal fibroblasts cells electroporation EGFP

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