CRISPR/Cas9介导的小鼠BRCA1/BRCA2/CDH1基因乳腺特异性修饰研究

Mammary tissue-specific modification of mouse BRCA1/BRCA2/CDH1 genes using CRISPR/Cas9

目的应用CRISPR/Cas9系统构建BRCA1、BRCA2和CDH1三基因乳腺特异性修饰小鼠模型。方法针对小鼠BRCA1、BRCA2和CDH1基因分别设计并合成sgRNA,构建p X330-sp Cas9-U6-gRNA共表达载体,将共表达载体中CBh启动子替换为乳腺组织特异性启动子WAP。利用小鼠受精卵原核注射的方法制备转基因小鼠。结果共注射受精卵190枚,得到活性受精卵130枚,移植4只受体鼠。产下F0代仔鼠42只,鉴定出11只转基因阳性小鼠,阳性率为26.19%。F0代阳性鼠繁育后得到39只F1代仔鼠,其中9只为F1代转基因阳性鼠。结论通过构建乳腺组织特异表达Cas9载体成功制备了转基因阳性鼠。

Objective To construct a mouse model with mammary tissue-specific modification of the BRCA1,BRCA2,and CDH1 genes using the CRISPR/Cas9 system. Methods Single-stranded RNAs（ sgRNAs） targeting the exons of the mouse BRCA1,BRCA2,and CDH1 genes were designed and synthesized,and the biological function of each sgRNA was validated in HEPA cells. Three sgRNAs were successively linked to the p X330-sp Cas9-U6-gRNA coexpression vector,and the CBh promoter was replaced by the mammary tissue-specific promoter WAP. The obtained final vector was linearized by the restriction endonuclease Sbf I,after which it was injected into mouse zygotes to generate transgenic mice.Results A total of 190 zygotes were injected and 130 positive zygotes were implanted into four recipient mice. There were42 offspring of F0 mice,while 11 mice were identified to have modified transgenes; the positive rate of transgenic mice was 26. 19%. A total of 39 F1 mice were obtained by F0 generation breeding and 9 F1 transgenic mice were detected.Conclusions Transgenic mice were successfully generated by constructing a mammary tissue-specific coexpression vector.