Trim26/ZNF173条件性基因敲除小鼠的制备

Establishment of Trim26/ZNF173 Conditional Gene Knockout Mice

摘要: 目的：制备Trim26/ZNF173条件性基因敲除小鼠，为研究Trim26基因在肝癌发生中的作用及其机制提供动物模型。方法：设计基因敲除策略，Trim26的外显子5~7缺失引起的移码突变会破坏蛋白结构域，进而导致Trim26蛋白功能丧失。为此，我们拟删除Trim26基因的外显子5~7，以借助Cre/lox P系统构建Trim26基因条件性敲除的小鼠。简单地说，先获得嵌合体小鼠，选择高嵌合率小鼠与B6/N小鼠交配获得F1代Trim26^fl/wt基因型的杂合子小鼠。F1代杂合子小鼠自交得到F2代Trim26^fl/fl基因型的纯合小鼠和对照野生型小鼠。提取鼠尾基因组DNA并行PCR鉴定基因型，在DNA水平确定小鼠的基因型。结果：成功构建了打靶载体，打靶载体经限制性内切酶线性化后，成功电转导入了B6/BLU ES 细胞中，ES克隆经过LR-PCR初筛及Southern blot进一步验证，确定得到了3株中靶ES细胞(即3D、6A和10F)，将中靶ES细胞扩增后进行囊胚注射，获得了12只Trim26的嵌合体小鼠(嵌合率不小于50%)。选择性成熟后的高嵌合率小鼠与B6/N小鼠交配繁育，得到F1代小鼠，PCR鉴定基因型确认获得了Trim26^fl/wt基因型的F1代杂合子小鼠。性成熟后的F1代杂合子小鼠合笼交配后得到F2代小鼠，PCR鉴定基因型确认获得了纯合的Trim26条件性基因敲除小鼠(基因型为Trim26^fl/fl)。结论：成功制备了Trim26条件性基因敲除小鼠，为进一步解析Trim26在肝癌发生中的作用及机制打下了良好基础。

Abstract: Objective: To establish the Trim26/ZNF173 conditional gene knockout mice model to lay the foun-dation for further research on the role of Trim26 gene in hepatocarcinogenesis and underlying mechanisms. Methods: Designing gene knockout strategies, the frame-shift mutation caused by the deletion of exon 5~7 of Trim26 will destroy the protein domain, which will lead to the function loss of Trim26 protein. Therefore, we plan to delete exons 5~7 of Trim26 gene to construct Trim26 gene conditional knockout mice with Cre/lox P system. In short, the chimeric mice were obtained first, and then the heterozygous mice (Trim26^fl/wt genotype) of F1 generation were obtained by mating the chimeric mice with B6/N mice. The F2 generation of homozygous mice with Trim26fl/fl genotype was obtained from F1 generation of heterozygous mice by inbreeding. Genomic DNA of mouse tail was extracted and the genotypes of mice were determined at DNA level by PCR. Results: The targeting vector was successfully constructed. The targeting vector linearized by restriction endonuclease was successfully electroporated into B6/BLU ES cells. The targeted ES cell clones were preliminarily screened by LR-PCR and further verified by Southern blot, and three targeted ES cells (3D, 6A and 10F) were obtained. By injecting the targeted ES cells into the blastocysts, 12 chimeric mice were obtained (the chimeric rate was not less than 50%). The F1 generation of heterozygous mice with Trim26^fl/wt genotype was obtained by mating of chimeric mice with B6/N mice. The F2 generation of Trim26 knockout mice with Trim26^fl/fl genotype were obtained by mating with F1 heterozygous mice with Trim26^fl/wt genotype. The genotypes of F1 and F2 generation mice were identified by PCR. Conclusion: Trim26 conditional gene knockout mice were successfully generated, which lays a good foundation for further understanding the roles of Trim26 in hepatocarcinogenesis and underlying mechanisms.