应用CRISPR/Cas9技术构建YOD1基因敲除小鼠

目的:应用CRISPR/Cas9技术构建去泛素化酶YOD1基因敲除小鼠。方法:针对YOD1基因设计单链向导RNA(sg RNA)识别序列,构建sg RNA质粒,与Cas9质粒体外转录、纯化后注射入受精卵,通过PCR和测序验证得到F0代阳性小鼠。配繁两代后,取同窝对照的野生型(WT)和敲除(KO)小鼠的主要组织器官研磨,使用免疫印迹(WB)技术检测各组织YOD1蛋白的表达,确证YOD1敲除小鼠模型是否成功建立。统计YOD1杂合子(HET)自交存活后代各基因型比例,分析是否有胚胎致死表型。解剖小鼠分析主要组织器官的表型,进一步利用H.E.染色分析KO小鼠是否存在自发的病理改变。通过血糖耐受实验(GTT)分析KO小鼠的血糖调控能力。结果:基因组测序和WB检测结果显示KO小鼠中YOD1被明显敲除,YOD1敲除小鼠模型成功建立。YOD1杂合子自交后代各基因型比例符合孟德尔定律,提示KO小鼠非胚胎致死。YOD1敲除小鼠肝脏显著小于WT小鼠。GTT结果表明敲除YOD1不影响小鼠的血糖稳态。结论:应用CRISPR/Cas9技术成功构建YOD1基因敲除小鼠。KO小鼠正常出生,无任何胚胎发育缺陷。与WT小鼠相比,KO小鼠肝脏显著减小,但无显著的自发病理变化,KO小鼠血糖控制亦无显著差异。

Construction of YOD1 Knockout Mice on CRISPR/Cas9 Technology

Objective: Construct YOD1 gene knockout mice based on CRISPR/Cas9 technology. Methods: Design and synthesize single-guide RNA (sgRNA) according to the YOD1 sequence in Genbank. Cas9 and sgRNA are transcribed to RNA in vitro, these RNA are then microinjected into zygotes of mice. The genotype is analyzed by PCR and sequencing. After YOD1 heterozygotes self-crossing and analysis of genotype of live offspring at weaning, wild type(WT)and knockout genotype(KO)littermates of YOD1 gene are verified. It is recorded that quantity and ratio of each genotype of live offspring of YOD1 heterozygotes self-crossing. And it is evaluated whether the ratio is in agreement with Mendel’s law of segregation. Protein lysates are made from main organs of the WT and KO littermates. And western blotting is used to assay the expression of YOD1 protein of these tissues. Meanwhile, size and weight of main organs and tissues of KO and WT mice are compared. Then analyze pathological phenotype of liver by H.E. staining. The glucose tolerance test (GTT) are carried out on the male mice of 6 months old. Results: According to PCR analysis and sequencing results, it is chose that mouse with deletion mutation and frameshift mutation in exon 2 of YOD1 gene to breed. After YOD1 heterozygotes self-crossing, WT and KO littermates are generated. According to statistics results, it is in agreement with Mendel’s law of segregation that the ratio of live offspring. Therefore, it is suggested that YOD1 KO mice birth normally without embryonic lethality. Western blotting results show that the expression of YOD1 in main organs is knocked-out significantly. Liver of YOD1 KO mouse is smaller in size than of WT littermate. There is no significant pathological phenotype in liver of YOD1 KO mice. YOD1 KO mice have general glycemic control in a GTT as compared to the control mice. Conclusions: YOD1 gene knockout mice are constructed successfully on CRISPR/Cas9 technology. And YOD1 KO mice birth and live normally without embryonic lethality. Compared to the control mice, livers of YOD1 KO mice are smaller in size and YOD1 KO mice have general glycemic control.