Construction of severe combined immunodeficiency mice based on CRSIPR/Cas9 technology
Received:May 06, 2016  
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DOI:10.3969/j.issn.1005-4847.2016.04.002
KeyWord:CRISPR/Cas9;Knockout;Severe combined immunodeficiency mice
                             
AuthorInstitution
赵亚 第四军医大学实验动物中心, 西安
李红武 北京艾德摩生物技术有限公司, 北京
师长宏 第四军医大学实验动物中心, 西安
张彩勤 第四军医大学实验动物中心, 西安
赵勇 第四军医大学实验动物中心, 西安
刘佩娟 第四军医大学实验动物中心, 西安
白冰 第四军医大学实验动物中心, 西安
唐娟 第四军医大学细胞工程中心, 西安
白杰英 军事医学科学院实验动物中心, 北京
张海 第四军医大学实验动物中心, 西安
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Abstract:
      Objective To knockout Rag2 and IL2rg 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 IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 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 mating, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion; however, there was only one genotype in Rag2 gene, which was 8 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 IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.
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