Constructing CXCR4 lentiviral vector and transfecting breast cancer cells
10.3969/j.issn.1006-5725.2015.17.007
- VernacularTitle:CXCR4慢病毒表达载体的构建及细胞转染
- Author:
Siqin DUAN
;
Yao FAN
;
Xue ZHAO
;
Yuanzhi CAO
;
Yi TAN
;
Beiguo LONG
;
Weifeng MA
- Publication Type:Journal Article
- Keywords:
CXCR4;
Lentivirus;
Breast cancer cells
- From:
The Journal of Practical Medicine
2015;(17):2786-2789
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct lentivirus containing CXCR4 gene and transfect MCF-7 cells , and obtain CXCR4 high-expressing MCF-7 cells. Methods CXCR4 gene was amplified by RT-PCR to construct CXCR4/pSin-EF2, which was transfected into HEK293T cells with psPAX2 and pMD2G vector for lentivirus packing. Packaged lentivirus was used to transfect human breast cancer cells MCF-7, with empty lentivirus as control. CXCR4 mRNA and protein expression levels were detected by RT-PCR and Western blot before and after transfection. And flow cytometry was used to detecte cell surface CXCR4 expression. Results The recombinant plasmid CXCR4/pSin-EF2 was constructed successfully,identified by double digestion and sequencing, and transfected into HEK293T cells to obtain high-titer lentivirus. RT-PCR and Western blot confirmed that the expression of CXCR4 in MCF-7 cells increased significantly after CXCR4 lentivirus transfection. Flow cytometry results showed that the CXCR4 positive rate increased from 26.78% to 99.29%, while there is no significant difference in CXCR4 expression between vector-transfected MCF-7 cells and non-transfected MCF-7 cells. Conclusion CXCR4 lentivirus and the breast cancer cell line with high and stable expression of CXCR4 (MCF-7CXCR4) were successfully constructed.
