生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 273-280.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1564

• 研究报告 • 上一篇    下一篇

基于CRSIPR/Cas9技术构建凝血因子8基因敲除小鼠模型及表型验证

王海杰(), 王成稷, 郭洋, 王云, 陈艳娟, 梁敏, 王珏, 龚慧, 沈如凌()   

  1. 上海实验动物研究中心,上海 201203
  • 收稿日期:2022-02-21 出版日期:2022-10-26 发布日期:2022-11-11
  • 作者简介:王海杰,男,实验师,研究方向:模式生物表型;E-mail: wanghaijie@slarc.org.cn

Construction of Coagulation Factor 8 Gene Knockout Mouse Model Based on CRSIPR/Cas9 Technique and Verification of Phenotype

WANG Hai-jie(), WANG Cheng-ji, GUO Yang, WANG Yun, CHEN Yan-juan, LIANG Min, WANG Jue, GONG Hui, SHEN Ru-ling()   

  1. Shanghai Laboratory Animal Research Center,Shanghai 201203
  • Received:2022-02-21 Published:2022-10-26 Online:2022-11-11

摘要:

基于CRISPR/Cas9技术构建凝血因子8(F8)基因敲除小鼠模型并对其表型进行初步验证。针对F8基因外显子1非编码区和外显子26下游序列设计sgRNA靶位点,体外转录获得sgRNA,与编码Cas9的mRNA混合后通过受精卵显微注射方法获得F0代阳性敲除小鼠,通过繁育及基因型鉴定获得F8基因敲除纯合子小鼠(F8 -/-小鼠);通过逆转录PCR(RT-PCR)检测主要组织中F8基因在mRNA水平的表达情况,通过酶联免疫吸附测定(ELISA)检测血浆中F8基因在蛋白水平的表达情况;通过活化部分凝血活酶时间检测(APTT)和纤维蛋白原含量(FIB)测定实验检测F8基因敲除后小鼠凝血功能情况。PCR及测序结果表明F8基因在小鼠基因组中被成功敲除;RT-PCR 和ELISA检测结果显示,F8 -/-小鼠中F8在mRNA和蛋白水平上表达均显著低于野生型小鼠(WT小鼠)(P<0.000 1,P<0.01);APTT、FIB和滴血实验检测结果显示,F8 -/-小鼠血浆凝固时间显著高于WT小鼠(P<0.05),F8 -/-小鼠存在严重凝血障碍表型,给予人凝血因子Ⅷ后凝血可恢复到正常水平。利用 CRISPR/Cas9技术成功构建F8基因敲除小鼠模型并初步验证其表型,证实该小鼠F8基因的缺失可以导致凝血功能障碍。

关键词: 凝血因子8(F8), CRISPR/Cas9, 基因敲除, 血友病A

Abstract:

A coagulation factor 8(F8)gene knockout mouse model was constructed based on CRISPR/Cas9 technique and its phenotype was verified. sgRNA target sites were designed according to exon 1 non-coding region to exon 26 downstream sequences of F8 gene,the sgRNA was obtained from in vitro transcription and mixed with mRNA of encoding Cas9. F0 generation positive knockout mice were obtained by microinjection of fertilized eggs. F8 -/- gene knockout homozygous mice(F8-/- mice)were obtained by breeding and genotype identification. The expression of F8 gene at mRNA level in main tissues was detected by reverse transcription PCR(RT-PCR),the protein expression of F8 gene in plasma was detected by enzyme-linked immunosorbent assay(ELISA),and the coagulation function of F8 knockout mice was detected by activated partial thromboplastin time(APTT)and fibrinogen content(FIB). PCR and sequencing results showed that F8 gene was knocked out successfully in mouse genome,and RT-PCR and ELISA results demonstrated that the expression of F8 in F8-/- mice was significantly lower than that in wild-type mice(WT mice)(P < 0.0001,P<0.01). APTT,FIB and drop blood test results revealed that the plasma coagulation time of F8-/- mice was significantly longer than that of WT mice. F8-/- mice had severe coagulation disorder phenotype,which restored to normal levels after administration of human factor VIII. The F8 gene knockout mice model is successfully constructed by CRISPR/Cas9 technique and its phenotype is preliminarily verified,which confirms that the deletion of F8 gene can lead to coagulation dysfunction.

Key words: coagulation factor Ⅷ(F8), CRISPR/Cas9, gene knockout, hemophilia A