Construction of human ARF4 lentiviral vector and stable expression in ovarian cancer cell line SKOV3
10.3760/cma.j.issn.1673-4181.2017.06.002
- VernacularTitle:人ARF4慢病毒表达载体的构建及其在卵巢癌细胞系SKOV3中的稳定表达
- Author:
Yimin ZHANG
1
;
Qihui WU
;
Xiaolei REN
;
Guang SHU
;
Jing WANG
;
Junyun CHENG
;
Ying WANG
;
Xinmei CAI
;
Shan ZHOU
;
Zhenbao LIU
;
Gang YIN
Author Information
1. 中南大学湘雅药学院
- Keywords:
Gene cloning;
Lentiviral vector;
ADP-ribosylation factor 4;
SKOV3 cell line
- From:
International Journal of Biomedical Engineering
2017;40(6):410-415,420
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer.
