内蒙古白绒山羊VEGF164基因毛囊特异性表达载体的构建及胎儿成纤维细胞的稳定转染

doi:10.3864/j.issn.0578-1752.2011.22.025

Abstract

Abstract: 【Objective】 The present study aims at constructing a eukaryotic expression vector pCDsRed2-KV of goat VEGF164 (vascular endothelial cell growth factor) gene and then to transfer it into Inner Mongolia Cashmere Goat (Capra hircus) fetal fibroblast (GFb) cells to obtain a transgenic cell clones, which stably expresses red fluorescence and expresses VEGF164 in hair follicle cells specifically. The transgenic cell clones can be used for nuclear transplantation. 【Method】 pCDsRed2-KV (6.3 kb), a hair-follicle-specific expression vector of VEGF164, was constructed by connecting VEGF164 gene to downstream of KAP6-1 promoter, and then inserting the KAP6-1 promoter-VEGF164 gene fragment into the basic vector pCDsRed2, which contains a DsRed expression unit. The Inner Mongolia Cashmere Goat fetal fibroblast (GFb) cells were transfected with the expression vector by lipofectamineTM2000. Transgenic cell clones were obtained after screening by G418. The recombinant of exogenous DNA was identified by polymerase chain reaction. 【Result】 The sequencing result showed that the VEGF164 gene was connected properly to the downstream of pKAP6-1, then the CMV promoter and the DsRed2 gene in sequence. Exogenous DNA in the cell clones was examined by PCR and the promoter KAP6-1 as well as VEGF164 gene has been integrated into GFb cells genome stably. 【Conclusion】 A hair-follicle-cell-specific expression vector of VEGF164 gene was constructed successfully and transfered into GFb cells. These data provide a way to obtain the transgenic goat by nuclear transfer in the future.

Key words: Inner Mongolia Cashmere Goat, VEGF, hair-follicle-cell-specific expression vector, stable transfection

BAO Wen-Lei, LI Bin, HOU Xin, LIU Jun-E, GUO Xu-Dong, WANG Zhi-Gang, LIU Dong-Jun. Construction of a Hair-Follicle-Cell-Specific Expression Vector of Goat VEGF164 Gene and Its Transfection into Caprine Fetal Fibroblasts Cells Stably[J].Scientia Agricultura Sinica, 2011, 44(22): 4756-4762.

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Construction of a Hair-Follicle-Cell-Specific Expression Vector of Goat VEGF164 Gene and Its Transfection into Caprine Fetal Fibroblasts Cells Stably

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