基于CRISPR/Cas9技术构建严重联合免疫缺陷小鼠  

Construction of severe combined immunodeficiency mice based on CRSIPR/Cas9 technology

作　　者：赵亚[1] 李红武 师长宏[1] 张彩勤[1] 赵勇[1] 刘佩娟[1] 白冰[1] 唐娟[3] 白杰英[4] 张海[1] 

机构地区：[1]第四军医大学实验动物中心,西安710032 [2]北京艾德摩生物技术有限公司,北京101111 [3]第四军医大学细胞工程中心,西安710032 [4]军事医学科学院实验动物中心,北京100071

出　　处：《中国实验动物学报》2016年第4期339-343,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：军队重点研究课题(NO:BWS14J058);国家自然科学基金面上项目(NO:81272385);陕西省科技资源统筹项目(NO:2014FWPT-11)

摘　　要：目的应用CRISPR/Cas9技术靶向敲除编码小鼠T、B细胞的Rag2基因及编码NK细胞的IL2rg基因,构建T、B细胞及NK细胞联合免疫缺陷小鼠。方法根据Genbank报道的Rag2及IL2rg基因序列,分别针对其外显子设计25 bp左右的sgRNA并进行合成,sgRNA退火后克隆入p X330载体。Rag2-sgRNA、IL2rg-sgRNA及Cas9重组质粒体外转录为mRNA后显微注射入BALB/c小鼠受精卵细胞,受精卵细胞移植到受体动物获得子代小鼠,首建鼠(F0)与野生型小鼠交配获得F1代小鼠,突变的F1代小鼠互交后筛选F2代纯合子小鼠。通过基因测序、流式细胞技术及接种人源性肿瘤细胞系方法检测子代小鼠基因型和表型。结果成功构建了Rag2-sgRNA、IL2rg-sgRNA重组质粒并对其进行了体外转录,mRNA显微注射并移植后获得57只F0小鼠。连续交配后,获得F2代纯合子小鼠。序列分析表明子代小鼠中IL2rg有两个基因型,分别是10 bp和11 bp的缺失突变;而Rag2只有一个基因型,为8 bp的缺失突变。与野生型BALB/c小鼠相比,小鼠外周血中CD3、B220及NKp46阳性细胞数量明显降低。接种人乳腺癌细胞系SKBR-2HL后,肿瘤生长良好,且随着时间延长肿瘤组织逐渐增大。结论利用CRISPR/Cas9技术可有效实现BABL/c小鼠体内Rag2、IL2rg基因突变,并导致小鼠T、B及NK细胞功能异常。Objective To knockout Rag2 and IL2 rg genes and construct severe combined immunodeficiency mice based on CRISPR / Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2 rg sequences in Genbank. After annealing,sgRNA was cloned into pX 330 vector. Recombination plasmid Rag2-sgRNA,IL2rg-sgRN and Cas9 were then transcribed into RNA,these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing,flow cytometry and xenograft model. Results Rag2-sgRNA and IL2rg-sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat-ing,F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2 rg gene,10 bp or 11 bp deletion; however,there was only one genotype in Rag2 gene,which was8 bp deletion. Compared with wild-type BALB / c mice,the number of CD3＋,B220＋and NKp46＋cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR-2HL cells,tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR / Cas9 is an efficient way to mutate Rag2 and IL2 rg gene in mice in vivo,leading to aberrant T cells,B cells and NK cells.

关 键 词：CRISPR/Cas9 基因敲除 免疫缺陷小鼠 

分 类 号：Q95-33[生物学—动物学]