Construction of lentiviral vector for truncated mouse fibroblast growth factor receptor-1 gene and its expression in eukaryotic cells.
- Author:
Wei CHEN
1
;
Chong CHEN
;
Huan-Xin ZHANG
;
Zhi-Ling YAN
;
Hai CHENG
;
Ling-Yu ZENG
;
Kai-Lin XU
Author Information
1. Department of Hematology, Xuzhou Medical College Affiliated Hospital, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
Genetic Vectors;
Humans;
Lentivirus;
genetics;
Mice;
Mice, Inbred BALB C;
Plasmids;
Receptor, Fibroblast Growth Factor, Type 1;
genetics;
Recombinant Fusion Proteins;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2012;20(1):168-172
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to clone the gene coding mouse fibroblast growth factor receptor-1 (fgfr1), to construct the recombinant lentiviral vector of truncated form fgfr-1 (Δfgfr1) carrying enhanced green fluorescence protein (EGFP) and to investigate its expression in eukaryotic cells (293FT cells). The full length fgfr1 gene was cloned by RT-PCR using brain tissue of BALB/c fetal mouse as template and inserted into PCR-Blunt vector, a truncated fgfr1 fragment was produced by site-directed mutagenesis for deleting intracellular phosphorylated domain, then was subcloned into a lentiviral vector and cotransfected into 293FT packaging 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 truncated FGFR1 protein was detected by Western blot. The results demonstrated that mouse fgfr1 gene was cloned and the lentiviral expression vector LV-IRES-EGFP-Δfgfr1 and control vector LV-IRES-EGFP were 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 293FT cells post transfection, and the transfection efficacy was > 95% determined by FCM. Expression of FGFR1 protein detected by Western blot was significantly higher than that in control group. It is concluded that the truncated gene fgfr1 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.
