摘要/Abstract
摘要： 目的·构建反转录病毒小向导RNA（small guide RNA，sgRNA）表达载体，用于小鼠T细胞分化调控及其基因功能的研究。方法·表达短发夹RNA（short hairpin RNA，shRNA） 的反转录病毒载体（MSCV-LTR-miR30-PIG，LMP）经过Xho1和Sal1双酶切后，回收得到反转录病毒载体骨架。以慢病毒sgRNA表达载体为模版，通过PCR扩增获得U6-sgRNA-PGK-Puro-BFP片段。利用同源重组将U6-sgRNA-PGK-Puro-BFP片段组装进反转录病毒载体骨架。为了验证系统的有效性，设计靶向Il17a、Rorc和Irf4的sgRNA序列并进行克隆。分离Cas9转基因小鼠CD4 T细胞分别进行sgRNA反转录病毒感染，诱导其向Th17细胞分化。细胞因子染色，流式检测Il17a阳性细胞比例。2组独立样本数据比较采用非配对t检验。结果·①成功构建反转录病毒sgRNA载体。②利用反转录病毒载体向小鼠CD4 T细胞递送sgRNA并实现Il17a基因敲除。③Rorc-sgRNA和Irf4-sgRNA阳性群体中，Il17a阳性群体比率显著降低（P<0.05）。结论·利用sgRNA反转录病毒载体成功向目的细胞递送sgRNA并实现基因敲除。Rorc和Irf4基因敲除结果提示该系统可用于小鼠T细胞基因功能研究。
关键词: CRISPR/Cas9, Cas9转基因小鼠, 反转录病毒载体, Th17细胞分化
Abstract:
Objective · To construct retroviral small guide RNA (sgRNA) expression vector for studying gene function in mouse T cells. Methods · The short hairpin RNA (shRNA)-expressing retroviral vector MSCV-LTR-miR30-PIG (LMP) was digested with Xho1 and Sal1 to obtain the backbone of the retroviral vector. The lentiviral sgRNA vector was used as template for PCR amplification to obtain the U6-sgRNA-PGK-Puro-BFP fragment. The PCR fragment was then assembled into the retroviral vector backbone by homologous recombination. To verify the effectiveness of the system, sgRNA sequences targeting Il17a, Rorc and Irf4 were designed and cloned. CD4 T cells isolated from Cas9 transgenic mice were infected with individual sgRNA retrovirus and differentiated into Th17 cells. After cytokine intracellular staining, the percentage of Il17a positive population was detected by flow cytometry. Unpaired t test was used to compare the data of independent samples between the two groups. Results · ①The sgRNA retroviral vector was successfully constructed. ② The retroviral vector was used to successfully deliver sgRNA to CD4 T cells, which mutated Il17a gene. ③The percentage of Il17a positive population was significantly reduced in the Rorc-sgRNA and Irf4-sgRNA positive populations (P<0.05). Conclusion · The sgRNA retroviral vector is successfully used to deliver sgRNA to target cells. In addition, the results of Rorc and Irf4 gene knockout experiments prove that the system can be used for studying gene function in mouse T cells.
Key words: CRISPR/Cas9, Cas9 transgenic mouse, retroviral vector, Th17 cell differentiation
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