p35^(Nck5a)基因重复性打靶载体的构建和ES细胞基因打靶研究

Construction of Gene Targeting Vector for Duplicating p35^(Nck5a) Gene and Its Application in the Gene Targeting of ES Cells

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复，采用常规的分子克隆技术，构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞，经过Ｇ４１８和ＧＡＮＣ分组药物选择，获得２４５个 双药物抗性的细胞克隆，细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定，２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复，同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。

To duplicating the regulatory subunit P^35~Nck5a gene of mouse neuronal cdc2-like kinase in embryonic stem (ES) cells, about 12.2kb of PGDTV vector for p35Nck5a gene duplication was constructed. The linearized pGDTV vectors were transfected into ES cells by electroporation. 245 drug-resistant cell clones were obtained in both G418 and GANC medium and the frequency of surviving cells was 6.22 × 10-5. Two ES cell clones were identified to have duplicated the P35Nck5a gene by use of both PCR and genomic Southern blotting, and the frequency of homologous recombination is 5.08 × 10-7. The use of negative selection gene (HSV-tk) results in 7-fold increase at selection efficiency. This study lays the foundations of preparing mouse models of Alzheimer' s disease.