Construction of Lentiviral Expression Vector Carrying SOX9 Gene and Its Stable Expression in LNcap Cell
10.3969/j.issn.1003-4706.2018.02.002
- VernacularTitle:SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达
- Author:
Jiang WANG
1
;
Jun-Jun DUAN
;
Xiao-Yu ZHANG
;
Xiao JIA
;
Xiao-Bo WANG
;
Ying-Hong HE
Author Information
1. 大理大学基础医学院;云南省高校细胞生物学重点实验室
- Keywords:
SOX9;
Lentiviral vector;
LNcap cell;
Liposome tansfection
- From:
Journal of Kunming Medical University
2018;39(2):5-9
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9 . Methods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector pLVX-IRES-Puro. pLVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening. The mRNA level and protein level in the infected LNcap cell were detected by qRT-PCR and Western blot respectively. Results Restriction enzyme digestion and sequencing demonstrated that SOX9 cDNA was successfully cloned into pLVX-IRES-FLAG lentiviral vector. After the transfection to LNCaP cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 mRNA detected by qRT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclusion The recombinant lentiviral vector bearing human SOX9 cDNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.
