Lentivirus Mediated Construction of Stably Expressing FABP4 Fibroblasts Line from the Small-tailed Han Sheep

doi:10.7668/hbnxb.2018.01.019

Abstract

Abstract: The aim of the study is to construct a fibroblast of Small-tailed Han sheep which could stably express FABP4.Firstly,the primary fibroblasts of Small-tailed Han sheep were prepared by tissue culture,then the sheep FABP4 gene was cloned by chemical synthesis and the DNA fragment was sub-cloned into a lentiviral vector.After packaging,the lentivirus was affected with the primary fibroblasts of Small-tailed Han sheep and purified by puromycin.At last,the expression of FABP4 protein was identified by Western Blot.The results showed that after passing 2-3 times,the primary fibroblasts of Small-tailed Han sheep were obtained with high purity.The titer assay showed that no fluorescence was observed when the amount of 10^-4 μL virus stock solution was added in the 96-well plate, while the fluorescence could be observed when the amount of 10^-3 μL virus stock solution was added, indicating that the titer of lentivirus was above 1×10⁶ TU/mL. After infected by the lentivirus and purified by puromycin,green fluorescence could be observed in all cells.The Western Blot showed that FABP4 protein expression was undetectable in the control group, but FABP4 protein expression was clearly observed in the virus-infected group, which indicated that the stably expressing FABP4 fibroblasts from the Small-tailed Han sheep were constructed successfully.

Key words: FABP4 gene, Lentivirus, Small-tailed Han sheep, Primary fibroblasts

CLC Number:

S826
Q78

HUA Liushuai, WANG Jing, BAI Jie, ZHU Xiaoting, CHEN Fuying, YU Xiang, CHU Qiuxia, XIN Xiaoling, XU Zhaoxue, ZHAO Hongchang, WANG Eryao. Lentivirus Mediated Construction of Stably Expressing FABP4 Fibroblasts Line from the Small-tailed Han Sheep[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(1): 121-126. doi: 10.7668/hbnxb.2018.01.019.

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