GDF9慢病毒载体的构建及其在山羊原代成纤维细胞中的稳定表达

doi:10.16431/j.cnki.1671-7236.2017.09.004

Abstract

Abstract:

This study was aimed to constract growth differentiation factor 9 (GDF9) lentiviral vector,which was stably expressed in goat primary fibroblast. The CDS of sheep GDF9 gene was cloned by gene synthesis, the CDS was 1 362 bp, encoding 453 amino acids. After double enzyme digestion and ligation, the GDF9 fragment was sub-cloned into an empty lentiviral vector. The GDF9 lentiviral vector and the packaging plasmids were co-transfected into 293T cells, and the lentivirus with titer of 1×10⁶ TU/mL was obtained. The goat primary fibroblasts were transfected with GDF9 lentivirus, the red fluorescence could be observed in more than 60% of the cells, suggesting the prepared lentivirus had high infection efficiency to goat primary fibroblasts. After puromycin screening, all cells were able to observe red fluorescence. Real-time quantitative PCR analysis showed that the GDF9 expression level was higher than that of control group. The results indicated that the goat primary fibroblasts stably expressing GDF9 were successfully obtained. This results might lay a foundation for the function study of GDF9 and the goat germplasm resources innovation in the future.

Key words: GDF9 gene; lentivirus; goat; primary fibroblasts

CLC Number:

Q782

HUA Liu-shuai, WANG Jing, CHEN Fu-ying, XIN Xiao-ling, CHU Qiu-xia, FENG Ya-jie, XU Zhao-xue, WANG Er-yao. Construction of GDF9 Lentiviral Vector and Its Stable Expression in Goat Primary Fibroblasts[J]. , 2017, 44(9): 2566-2572.

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Construction of GDF9 Lentiviral Vector and Its Stable Expression in Goat Primary Fibroblasts

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