[Construction of HBD-3 gene mammary-specific expression vector and eukaryotic expression].
To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human beta-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5' and 3' regulating region of beta-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human beta-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human beta-defensin-3 in the supernatant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.
Peng
Wei
W
Institute of Biotechnology, College of Animal Veterinary Medicine, Northwest Agiculture and Forestry University, Yangling 712100, China.
Lan
Zhigang
Z
Ma
Jingjing
J
Wang
Baolei
B
Zhang
Yong
Y
D000818:Animals; D030801:Animals, Genetically Modified; D002364:Caseins / Q000235:genetics; D002417:Cattle; D004847:Epithelial Cells / Q000378:metabolism*; D018773:Genes, erbB-1 / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
D002364:Caseins; C422950:DEFB103A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins