Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (2): 260-264.doi: 10.3969/j.issn.2095-4344.1992

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Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination

Li Lei, Xie Jianshan, Du Jiazheng, Shi Liang, Cui Huilin#br#   

  1. Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Received:2019-03-11 Revised:2019-03-20 Accepted:2019-05-23 Online:2020-01-18 Published:2019-12-25
  • Contact: Cui Huilin, MD, Associate professor, Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Li Lei, Master, Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:
    the Shanxi Provincial Key Dominant Subject Project, No. 2011-2014; the Basic Medical Science and Technology Foundation of Shanxi Medical University, No. 201410; the Science and Technology Innovation Foundation of Shanxi Medical University, No. 01200719

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Abstract:

BACKGROUND: Lentiviral vectors have been widely used as exogenous transgenic vectors. However, a recombinant lentiviral vector containing rat diacylglycerol kinase γ (DGKγ) gene has not been reported.

OBJECTIVE: To construct lentiviral overexpression vector of rat DGKγ by homologous recombination.

METHODS: Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats, and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKγ gene CDS. Then, the two homologous recombination fragments were ligated into the plasmid vector. The positive clones were confirmed by PCR and DNA sequencing. The CMV-rat DGKγ-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells. The expression of GFP in infected 293T cells was observed under fluorescence microscope. Real-time PCR and western blot assay were used to detect the relative expression of DGKγ mRNA in infected 293T cells.

RESULTS AND CONCLUSION: The results of PCR simplification and sequencing indicated that the CMV-rat DGKγ-GFP lentiviral vector was successfully constructed. In 293T cells infected with CMV-rat DGKγ-GFP lentivirus, the expression of GFP was observed under fluorescence microscope and the DGKγ mRNA expression was increased significantly than that of the vector group by real-time PCR (P < 0.01). Western blot assay results showed that the DGKγ protein expression of the selected GFP-positive 293T cells was increased very significantly (P < 0.001). To conclude, the rat DGKγ lentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells. 

Key words: diacylglycerol kinase γ, DGKγ, homologous recombination, lentiviral vector, overexpression

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