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-...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.展开更多
基金supported by A key special project of breeding for disease resistance of PR china(Project No.2008ZX08007-004)
文摘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.
