Construction of lentiviral vector for mouse CXC chemokine receptor 4 gene and its expression in eukaryotic cells.
10.7534/j.issn.1009-2137.2013.01.040
- Author:
Miao LI
1
;
Wei CHEN
;
Xin-Jiang AN
;
Jun-Hua CAO
;
Cong-Hai FAN
;
Li-Yang YIN
;
Chun-Xia ZHANG
;
Hao-Miao SUN
Author Information
1. Department of Infection, Xuzhou Children's Hospital, Xuzhou, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
Gene Expression;
Genetic Vectors;
Green Fluorescent Proteins;
genetics;
Humans;
Lentivirus;
genetics;
Mice;
Mice, Inbred C57BL;
Plasmids;
Receptors, CXCR4;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2013;21(1):198-202
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to clone the gene coding mouse CXC chemokine receptor 4 (CXCR4), to construct the recombinant lentiviral vector carrying enhanced green fluorescence protein (EGFP) and to explore its expression in eukaryotic cells (293FT cells). The full length CXCR4 gene was cloned by RT-PCR using bone marrow cells from C57BL/6 mouse as template and inserted into PCR-Blunt vector. CXCR4 fragment was generated by digestion with restriction endonuclease and subcloned into a lentiviral vector to generate recombinant lentiviral vector LV-IRES-EGFP-CXCR4, which was co-transfected into 293FT cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM). And the expression of CXCR4 protein was detected by Western blot. The results demonstrated that mouse CXCR4 gene was cloned and the lentiviral vector was successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in the transfected 293FT cells, and the transfection efficacy > 95% was determined by FCM. Expression of CXCR4 protein detected by FCM and Western blot was significantly higher than that in control group. It is concluded that the CXCR4 gene along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.
