[1]李昂,王辉,邵玉乐,等.TRIM30α-/-小鼠模型的构建及其表型分析[J].中国预防兽医学报,2021,(02):191-197.[doi::10.3969/j.issn.1008-0589.202005014]
 LI Ang,WANG Hui,SHAO Yu-le,et al.Construction and phenotypic analysis of TRIM30α-/- mouse model[J].Chinese journal of preventive veterinary medicine,2021,(02):191-197.[doi::10.3969/j.issn.1008-0589.202005014]
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TRIM30α-/-小鼠模型的构建及其表型分析
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
191-197
栏目:
免疫学
出版日期:
2021-03-16

文章信息/Info

Title:
Construction and phenotypic analysis of TRIM30α-/- mouse model
文章编号:
:1008-0589(2021)02-0191-07
作者:
 李昂12王辉2邵玉乐2许曼2张子博2田璐2史鑫琪2孟庆文2*陈洪岩12*
 (1. 东北农业大学生命科学学院,黑龙江哈尔滨150030; 2. 中国农业科学院哈尔滨兽医研究所兽医生物技术
国家重点实验室/黑龙江省实验动物与比较医学重点实验室,黑龙江哈尔滨150069)
Author(s):
 LI Ang12 WANG Hui2 SHAO Yu-le2 XU Man2 ZHANG Zi-bo2 TIAN Lu2 SHI Xin-qi2MENG Qing-wen2* CHEN Hong-yan12*
 (1. College of Life Science, Northeast Agricultural University, Harbin 150030, China; 2. State Key Laboratory of Veterinary Biotechnology,
Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine/Harbin Veterinary Research Institute, Chinese
Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
:TRIM30αCRISPR/Cas9表型分析动物模型
Keywords:
: TRIM30α CRISPR/Cas9 phenotypic analysis animal model
分类号:
S852.4
DOI:
:10.3969/j.issn.1008-0589.202005014
文献标志码:
A
摘要:
: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。
Abstract:
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 TRIM30α gene, we used the CRISPR/Cas9 system to edit mouse TRIM30α gene 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 TRIM30α gene in mice (heart, liver, spleen, lungs, kidneys, brain, muscles) at the
DNA level was identified by PCR and sequencing; the transcription levels of TRIM30α gene 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 TRIM30α gene- 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 TRIM30α gene 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 TRIM30α in the natural immune response mediated by DNAviruses.

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更新日期/Last Update: 2021-03-17