Establishment of a method to effectively knock down gene expression in primary B cells to quickly identify functional gene
LIU Jia, WAN Simin, BAI Lu, LI Jianhua
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), and Department of medical microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Abstract：The process of B cell-mediated antiviral humoral immune response involves the up-regulation of genes. In order to identify the function of these genes quickly, we need a method to knock down target gene expression in B cells effectively in vitro and in vivo. In this study, four procedures were adopted to improve the assay. Firstly, we co-transfected the Drosha enzyme-specific siRNA with the retroviral packaging plasmid into virus packaging cells. Secondly, we constructed an in vitro culture system for primary B cell by adding anti-CD180 antibody to the culture medium, in which B cells can proliferate robustly. Then, by increasing the times of spin infection, the transduction efficiency of B cells was further improved. Besides, through pre-infection of mice, more proliferated and differentiated B cells after adoptive transfer can be harvested for phenotypic analysis. By adopting the above-mentioned improvement measures, the expression of B cell functional gene Bcl6 was successfully knocked down, and its anti-apoptotic function was verified. Establishment of this method would lay a good foundation for studying the mechanism of B cell proliferation, differentiation and defection in acute and chronic viral infections in the future.
刘佳，万思敏，白露，李建华. 一种有效敲低原代B细胞中目的基因表达以快速鉴定基因功能方法的建立[J]. 微生物与感染, 2020, 15(4): 220-227.
LIU Jia, WAN Simin, BAI Lu, LI Jianhua. Establishment of a method to effectively knock down gene expression in primary B cells to quickly identify functional gene. JOURNAL OF MICROBES AND INFECTIONS, 2020, 15(4): 220-227.
2020, Vol. 15 
