摘要
目的·通过优化后的CRISPR/Cas9基因编辑系统,研究原代B细胞中转录因子T-bet对抗体类别转换的调控作用。方法·系统性分析GEO公共数据库中体外诱导分化的B细胞转录组测序结果,探索转录因子T-bet对抗体类别转换的调控。构建靶向基因为Tbx21(T-box 21,编码T-bet)的小向导RNA(small guide RNA,sgRNA)反转录病毒表达载体,使用Plat-E细胞系作为病毒包装细胞系获得装有sg-Tbx21质粒的反转录病毒并对其浓缩。使用B细胞受体抗体、抗CD40抗体和Toll样受体激动剂体外协同刺激Cas9转基因小鼠的脾脏B细胞,感染高滴度反转录病毒颗粒实现基因编辑。流式细胞术分选感染细胞,提取基因组DNA进行PCR扩增后对靶向序列进行测序,利用ICE CRISPR分析工具计算基因敲除效率。体外诱导B细胞分化为浆细胞,采用ELISA检测基因编辑后B细胞培养上清液中IgM、IgG2c、IgG3抗体类别的丰度,通过独立样本t检验比较sg-NC组与sg-Tbx21组细胞上清液中抗体水平的差异。结果·①经分析GEO数据库发现,γ干扰素可以诱导B细胞中Tbx21基因的表达,且在T-bet的转录调控下,B细胞展现抗体类别转换相关通路活化的转录组特征。②使用浓缩后的高滴度病毒感染激活的B细胞,可以构建高效的反转录病毒感染原代B细胞体系。③Cas9转基因小鼠原代B细胞转染sg-Tbx21质粒后成功实现Tbx21基因敲除,敲除效率约为59%。④在体外诱导B细胞分化为浆细胞的条件下,sg-Tbx21转染后的B细胞培养上清液中IgG2c水平显著降低(P=0.000),IgM和IgG3的水平无明显的变化。结论·通过浓缩反转录病毒和协同刺激激活原代B细胞的方式可提高sgRNA递送的效率,在Cas9转基因小鼠原代B细胞中敲除Tbx21基因;应用这一系统证明了T-bet敲除后阻碍了IgG2c抗体类别的产生。
Objective·To study the regulation of transcription factor T-bet in antibody isotype switching via the optimized CRISPR/Cas9-mediated gene editing system in primary B cells.Methods·In order to demonstrate the regulation of T-bet in antibody isotype switching,the transcriptome data of in vitro-derived B cells from public database GEO was analyzed.The retroviral small guide RNA(sgRNA)vector was constructed to deliver sgRNA targeting Tbx21(T-box 21,encoding T-bet),and retroviral packaging cell line Plat-E was used for retrovirus production.The retroviruses with sg-Tbx21 plasmids were harvested and concentrated.Primary B cells isolated from Cas9 transgenic mice were activated in vitro by synergistic stimulation of anti-B cell receptor antibody,anti-CD40 antibody and a Toll-like receptor agonist,and then infected with concentrated retroviruses with sg-Tbx21.The genome DNA was extracted from the infected cells isolated by flow cytometry,and then PCR amplification was performed for target gene sequence.ICE CRISPR analysis tool was used to calculate the gene knockout efficiency.The edited B cells were induced to plasma cells in vitro and the antibody isotypes of IgM,IgG2c,and IgG3 production in the culture supernatant were assessed by ELISA.Independent sample t-test was used to analyze the difference of antibody levels in the supernatant between the sg-NC editing cells and sg-Tbx21 editing cells.Results·①According to the analysis of GEO database,interferon-γinduced the expression of Tbx21 in B cells,and T-bet expression promoted antibody isotype switching-related pathways.②The retroviral infection efficiency was improved in primary B cells after in vitro activation,which were infected by the concentrated high titer viruses.③The sg-Tbx21 plasmids were delivered to Cas9 transgenic B cells by retroviruses,which led to T-bet knockout efficiency of about 59%.④Cultured in the plasma cells-polarizing condition,the Tbx21 edited B cells secreted less IgG2c into the culture supernatant(P=0.000),while the levels of IgM and IgG3 did not significantly change.Conclusion·The efficiency of sgRNA delivery can be improved by concentrating retroviruses and activating primary B cells through synergistic stimulation,and Tbx21 gene can be knocked out in primary B cells of Cas9 transgenic mice;it is proved that T-bet knockout hinders the production of antibody isotype IgG2c through this system.
作者
韩夏夏
顾霜霜
戴黛
沈南
HAN Xiaxia;GU Shuangshuang;DAI Dai;SHEN Nan(Department of Rheumatology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai Institute of Rheumatology,Shanghai 200127,China)
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2022年第4期433-442,共10页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(31630021,31930037,82071843,81901637)
上海交通大学医学院高水平地方高校创新团队(SSMUZDCX20180100)。
