髓系特异性Spi1基因敲除小鼠的构建和基因鉴定

Construction and gene identification of myeloid-specific Spi1 knockout mice

目的构建髓系特异性Spi1基因敲除小鼠并分析其基因型,为疾病病理机制及药物靶点研究提供动物模型基础。方法根据CRISPR/Cas9技术原理和Cre/LoxP系统,设计并构建sgRNA和Donor载体,以第2号外显子(Exon 2)所在的转录本为敲除区域,在Exon 2两侧各放置同向Loxp元件;将Cas9蛋白、sgRNA和Donor载体混合显微注射到C57BL/6J小鼠的受精卵中,移植受精卵到假孕的C57BL/6J母鼠的子宫中,19~20 d后获得F0代。将阳性F0代小鼠与C57BL/6J小鼠交配,得到稳定的F1代Spi1^(flox/+)小鼠。F1代Spi1^(flox/+)小鼠雌雄自交得到Spi1^(flox/flox)小鼠。Spi1^(flox/flox)与Lyz2-Cre^(+)小鼠交配得到Spi1^(flox/+)/Lyz2-Cre^(+)小鼠,再将其与Spi1^(flox/flox)交配,得到的Spi1^(flox/flox)/Lyz2-Cre^(+)小鼠为髓系特异性Spi1基因敲除(KO)小鼠;Spi1^(flox/flox)/Lyz2-Cre^(-)小鼠作为野生型(WT)小鼠。提取WT和KO小鼠DNA,PCR扩增后琼脂糖凝胶电泳鉴定基因型;Western blot检测WT和KO小鼠免疫细胞中脾病灶形成病毒前病毒整合癌基因-1/富含嘌呤盒1(PU.1)表达。结果PCR鉴定结果显示,采用flox引物鉴定时仅扩增出220 bp条带的小鼠,即基因型为Spi1^(flox/flox)纯合子,采用Lyz2-Cre引物鉴定时扩增出700 bp的小鼠,基因型为Lyz2-Cre^(+);Western blot结果显示,与WT组比较,KO组小鼠骨髓来源巨噬细胞(BMDMs)和腹腔原位巨噬细胞(PM)中的PU.1不表达(P<0.01);T细胞中KO小鼠PU.1表达水平与WT小鼠差异无统计学意义;PCR和Western blot结果均表明髓系特异性Spi1 KO小鼠构建成功。结论成功构建和鉴定髓系特异性Spi1 KO小鼠,为进一步揭示PU.1在免疫调节中的潜在机制研究提供动物模型基础。

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods According to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were designed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19~20 days.Positive F0 mice were mated with C57BL/6J mice to obtain stable F1 Spi1^(flox/+)mice.Spi1^(flox/+)mice of F1 generation were selfed to obtain Spi1 ^(flox/flox) mice.Spi1^(flox/flox) mated with Lyz2-Cre+mice to obtain Spi1^(flox/+)/Lyz2-Cre^(+)mice,and then mated with Spi1 ^(flox/flox),the Spi1^(flox/flox)/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1^(flox/flox)/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electrophoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1^(flox/flox) homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre^(+).Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macrophages(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and Western blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-specific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.