小鼠PKD1基因的克隆及打靶载体的构建

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－Ｎ

Cloning of mouse PKD1 gene and constructing a knockout vector

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.