Construction of lentiviral vectors of fibronectin type Ⅲ domain-containing protein 5 overexpression and stable transfection of THP-1 cell line

Abstract:

Objective：To construct the lentiviral vector of fibronectin type Ⅲ domain-containing protein 5 （FNDC5） overexpression，and to obtain the THP-1 cell line stably transfected with FNDC5. Methods：FNDC5 gene was amplified by PCR and was then inserted to pLenti-EF1a-EGFP-P2A-Puro lentiviral vector. The recombinant vector was amplified in DH5α competent cells，and after se－quencing and identification，the vectors were transfected into 293T cell with the 4-plasmid packaging system to produce overexpression lentiviral particles. Virus titer was then measured. Western blot was used to measure the expression of FNDC5 in recombinant lentivirus；the recombinant lentivirus was transfected into THP-1 cell line，and a fluorescence microscope was used to calculate trans－fection efficiency. After a stably transfected cell line with FNDC5 overexpression was screened out by puromycin，the cells were divid－ed into blank group，empty group，and FNDC5 group，and Western blot and RT-PCR were used to measure the protein and mRNA expression of FNDC5. Oil red O staining was used to observe the effect of FNDC5 overexpression on lipid accumulation in THP-1 macrophage-derived foam cells. Results：PCR product identi－fication and sequencing results showed that a lentiviral vector of FNDC5 overexpression was successfully constructed. Green flu－orescent protein was detected after transfection of 293T cells，and the Flag-tag expression of FNDC5 was detected by Western blot. The titer of the lentivirus after packaging was 1.32×109 TU/mL. Western blot and RT-PCR were used to analyze the THP-1 cell line transfected with the lentivirus；compared with the empty group，the FNDC5 overexpression group had a significant increase in the protein and mRNA expression of FNDC5（238.0±24.9）% and （228.5±3.4）%，respectively，both P=0.000）. There was a reduction in lipid accumulation in THP-1 macrophage-derived foam cells with FNDC5 overexpression. Conclusion：A lentiviral vector of FNDC5 overexpression is successfully constructed in this study and a THP-1 cell line stably transfected with FNDC5 is established by the lentiviral vector of FNDC5 overexpression，indi－cating that FNDC5 can significantly reduce lipid accumulation in THP-1 macrophage-derived foam cells，which lays a foundation for further research.