CD4^(+)T细胞诱导性缺失小鼠模型构建

Generation of a mouse model for inducible depletion of CD4^(+)T cells

目的构建白喉毒素诱导的CD4^(+)T细胞特异性缺失小鼠(Cd4-DTR)模型。方法利用CRISPR/Cas9技术,通过同源重组的方式,在C57BL/6小鼠Cd4基因终止密码子位点定点插入IRES-DTR表达元件。流式细胞术检测未经处理的Cd4-DTR小鼠脾和硬脑膜CD4^(+)T细胞数量及比例与野生小鼠有无差异,单次注射不同剂量白喉毒素后CD4^(+)T细胞的减少程度以及CD4^(+)T细胞缺失的持续时间。结果鼠尾PCR鉴定证明Cd4-DTR模型构建成功,未经处理的Cd4-DTR小鼠脾中的CD4^(+)T细胞数量及比例低于野生小鼠,但硬脑膜中没有差异。单次注射75 mg/kg白喉毒素后Cd4-DTR小鼠的脾和硬脑膜中的CD4^(+)T细胞数量及比例都大幅度下降且都有显著性差异(P<0.01),单次白喉毒素注射后脾脏中的CD4^(+)T细胞缺失持续时间超过1周。利用3%DSS诱导小鼠急性结肠炎,结果显示Cd4-DTR小鼠肠道的病理改变弱于对照小鼠。结论成功构建Cd4-DTR小鼠模型,为CD4^(+)T细胞功能研究提供了新的小鼠模型。缺失CD4^(+)T细胞可能减轻DSS诱导的小鼠急性结肠炎。

This study aimed to establish a mouse model with diphtheria toxin-induced depletion of CD4^(+)T cells(referred to as Cd4-DTR mouse)by CRISPR/Cas9 technology,that is,IRES-DTR segment was inserted at the stop codon of Cd4 gene by homologous recombination.The proportion and number of CD4^(+)T cells in the spleen and cranial dura were detected by flow cytometry.Data showed that the number and proportion of CD4^(+)T cells in the spleen and cranial dura decreased significantly after Cd4-DTR mice were injected with 75 mg/kg diphtheria toxin.In the spleen,CD4^(+)T cell ablation could last more than 1 week after a single diphtheria toxin injection.And we found that the colon pathological changes were milder in Cd4-DTR mice with acute colitis induced by 3%DSS.So,deletion of T cells may have a protective effect on DSS-induced acute colitis.Therefore,Cd4-DTR murine model has been constructed successfully,which provides a new mouse model for the study of CD4^(+)T cell function.