小鼠PKD1基因的克隆及打靶载体的构建

Cloning of mouse PKD1 gene and constructing a knockout vector

目的：克隆小鼠的多囊肾病１（ＰＫＤ１）基因，构建ＰＫＤ１基因的Ｋｎｏｃｋｏｕｔ载体，为产生ＰＫＤ１基因缺陷小鼠品系准备条件。方法：以正常小鼠基因组ＤＮＡ为模板，扩增小鼠ＰＫＤ１基因部分序列为探针，应用噬斑原位杂交技术筛选１２９ＳｖＴｅｒ小鼠基因组ＤＮＡ文库，并应用Ｓｏｕｔｈｅｒｎ杂交、亚克隆及测序等方法鉴定所得克隆。对克隆到的ＰＫＤ１基因组ＤＮＡ片段进行结构分析，选择合适的亚克隆片段，以ｐＴＫ－ＮＥＯ质粒为框架构建目标载体。结果：筛选得到１个阳性克隆，证实了所得序列与基因库收录的序列一致，并成功构建了符合设计要求的目标载体。结论：克隆到含有ＰＫＤ１基因目的区域的基因组ＤＮＡ片段以及目标载体的成功构建，为建立小鼠ＰＫＤ１胚胎干细胞基因打靶动物模型奠定了基础。

Objective: To obtain mouse polycystic kidney disease 1 (PKD1) gene from a genomic library and construct a knockout vector. Methods: T Using partial mouse PKD1 genomic DNA fragments amplified by PCR as probes, a genomic library of 129SvTer mouse in bacteriophage vector was screened by plagues in situ hybridization .The inserts of genomic DNA fragments was analyzed by Southern blot, subclone and verified by sequencing. And then 2 fragments were chosen to clone into the pTKNEO plasmid. Results: (1) A positive phage clone was obtained after fourround screening. The sequence of the genomic DNA fragments inserted in the positive clone was in accordance with that of the Genebank. (2) A knockout vector was successfully constructed. Conclusion: This work is beneficial for making mice model of PKD1 gene knockout.