[Construction and application of retroviral vector system useful for high-efficiency transfection into human T cells]

Abstract

Aim: To construct a retroviral vector system with high-efficiency transfection into human T cells and carrying a rapid screening label, and compare it with the traditional retroviral vector system.

Methods: Retroviral vector pCMMP-IRES-GFP was first constructed by inserting the internal ribosome entry site-green fluorescent protein (IRES-GFP) cDNA into retroviral vector pCMMP. The chimeric TCR gene was inserted into pCMMP-IRES-GFP and then co-transfected into packaging cell line 293T with other two assistant vectors pMD. MLVgag. pol and pHDM. G. After 48 h, the culture supernatant was harvested and condensed by centrifugation. Meanwhile, the chimeric TCR gene was inserted into pLXSN and then transfected into packaging cell line PA317. The transfected PA317 cells were obtained by G418 pressure screening. The cells culture supernatant containing viruses was harvested after being cultured for 48 h. The viral titer was determined by NIH3T3 cells infection. The preactivated primary human T lymphocytes were infected by appropriate volume of viral fluid and detected by fluorescent microscopy or flow cytometry after 48 h.

Results: A retroviral vector pCMMP-IRES-GFP was constructed successfully. Compared with the traditional vector system, the viral titer was 2.15x10(11) VP/L vs 6.43x10(9) VP/L, and efficiency of transfection into preactivated primary T cells was 50%-60% vs 5%-10%. Furthermore, the infected cells were also detected by fluorescent microscope and could sorted by fluorescent activated cell sorting.

Conclusion: A retroviral vector system with high transfection efficiency into T lymphocytes and carrying a rapid screening label has been constructed, which establishes the foundation for basic and clinical studies on T lymphocytes.