TRIM30α^(-/-)小鼠模型的构建及其表型分析

Construction and phenotypic analysis of TRIM30α^(-/-) mouse model

TRIM30α作为TRIM蛋白家族成员,在天然免疫系统中发挥重要作用。为了研究TRIM30α基因的特性及功能,本研究在小鼠TRIM30α基因第一段编码区选择一段作为靶序列,合成了2条sgRNA,构建了重组质粒pUC19-TRIM30α-sgRNA1/2并体外转录后分别与体外转录并加poly(A)尾的pT7-SpCas9-SV40混合后经显微注射至C57BL/6近交系小鼠的受精卵中,获得第一代(F0代)TRIM30α基因敲除的小鼠并经PCR鉴定。将其与野生型(WT)小鼠合笼繁育出TRIM30α^(+/-)小鼠(F1代),经PCR鉴定后再通过全同胞交配的方式获得TRIM30α基因敲除的纯合子(TRIM30α^(-/-))小鼠(F2代)。通过PCR和测序在DNA水平鉴定TRIM30α^(-/-)小鼠各组织(心、肝、脾、肺、肾、脑、肌肉)TRIM30α基因的敲除情况;经RT-PCR检测TRIM30α^(-/-)小鼠上述各组织TRIM30α基因mRNA的转录水平;利用PCR分别扩增小鼠中的3个高频脱靶位点(Trim30d、D6WSu163e、Sbk1)后经T7E1酶切检测TRIM30α^(-/-)小鼠的脱靶效应。结果显示,分别获得了缺失了4 bp和7 bp片段的两种TRIM30α基因缺失的F0代小鼠(TRIM30α^(+/-)),选择缺失7 bp的TRIM30α^(+/-)小鼠繁育获得F1、F2代小鼠。PCR鉴定结果显示,共获得了7只TRIM30α^(-/-)纯合子小鼠;从DNA水平和mRNA转录水平鉴定结果显示,TRIM30α^(-/-)小鼠各组织的基因组DNA扩增条带均小于WT小鼠的扩增条带;且其体内未检测到TRIM30α基因的mRNA;TRIM30α^(-/-)小鼠体内也未检测到其他位点的脱靶编辑;TRIM30α^(-/-)小鼠的生长发育、血常规、血生化指标与WT小鼠相比均无显著差异。以上结果表明TRIM30α^(-/-)小鼠模型构建成功。本研究为探究DNA病毒介导的天然免疫应答中TRIM30α所发挥的作用提供了实验材料和研究依据。

As a member of the TRIM protein family,TRIM30α plays an important role in the innate immune system.To study the characteristics and functions of TRIM30αgene,we used the CRISPR/Cas9 system to edit mouse TRIM30αgene by targeting the first exon of the target gene.The Cas9 plasmid and the in vitro transcription product containing target sequence of sgRNA were microinjected into the fertilized eggs of C57BL/6 mice to obtained the first generation of knockout mice(F0),which were identified by PCR.The F0 TRIM30α^(-/-)mice were then cohabited with wild-type(WT)mice to generate TRIM30α^(+/-)mice(F1),which were confirmed by PCR.The TRIM30α^(+/-)mice were inbred by means of full-sib mating to generate the F2 offspring(TRIM30α^(-/-)mice).The knockdown efficacy of TRIM30αgene in mice(heart,liver,spleen,lungs,kidneys,brain,muscles)at the DNA level was identified by PCR and sequencing;the transcription levels of TRIM30αgene were also detected by RT-PCR in mice;and the off-target effects of CRISPR in TRIM30α^(-/-)mice were analyzed the amplicons of 3 high frequency off-target points Trim30d,D6WSu163e and Sbk1 by T7E1 enzymatic digestion.The results showed that two types of TRIM30αgene-editing F0 mice(TRIM30α^(+/-))were generated with either 4 nucleotides or 7 nucleotides deletion,and TRIM30^(+/-)mice with 7 nucleotides were selected for F1 and F2 generation mice.The PCR results showed that a total of 7 TRIM30α^(-/-)homozygous mice were generated.The PCR results based on genomic DNA showed that the genomic DNA amplification in TRIM30α^(-/-)mouse tissues was all smaller than that in WT mice,the transcription level of TRIM30αgene was not detected in TRIM30α^(-/-)mice;off-target editing was negative in TRIM30α^(-/-)mice;the growth and development,blood routine examination and biochemical indexes of TRIM30α^(-/-)mice were not significantly different from those of WT mice.The TRIM30α^(-/-)mice was successfully generated.This study provides the experimental materials and research basis for further elaborating the role of TRIM30αin the natural immune response mediated by DNAviruses.