摘要
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.
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-Iprl The mouse Iprl 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 culotte （4th passage） were transfected with pSRA-EGFP-Iprl by electroporation. Fluorescence from GFP was observed after 24 h. Transgenic cells were selected using G418 and the resultant monoclones were picked and expanded. The transgenic cells, at the 9th passage, were evaluated by PCR and flow cytometry. The inserted Iprl 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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2011年第B12期7-12,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
supported by A key special project of breeding for disease resistance of PR china(Project No.2008ZX08007-004)
