含小鼠糖皮质激素受体第二外显子基因片段的克隆和结构分析

CLONING AND STRUCTURE ANALYSIS OF GENE FRAGMENT BEARING MOUSE GLUCOCORTICOID RECEPTOR GENE EXON Ⅱ

克隆适于构建可调控基因打靶载体的小鼠糖皮质激素受体（ＧＲ）基因片段，为建立糖皮质激素受体基因缺陷型小鼠模型奠定基础。用ＰＣＲ扩增小鼠ＧＲ基因第二外显子上５６２ｂｐ的核酸片段作为探针，筛选小鼠基因组文库，共获得６个阳性克隆。对Ｃ１０克隆进行详细的测序和酶切图谱和Ｓｏｕｔｈｅｒｎ杂交分析后获得一６．５ｋｂ的基因片段。该片段含完整的ＧＲ基因第二外显子，左右分别有３９ｋｂ和１４ｋｂ的ＤＮＡ片段可作为下一步构建基因打靶载体的同源臂。

To clone a genomic DNA fragment containing mouse glucocorticoid receptor (GR) gene for the construction of an inducible gene targeting vector used in the establishment of a GR gene dificient mouse model. The mouse genomic library was screened by using a 562bp PCR product from GR gene exon Ⅱ as a probe. Six positive clones were got after screening. The DNA of positive clones was profoundly characterized by sequencing, restriction mapping and Southern blotting to get the right fragment. A 6.5 kb DNA fragment was separated from the C10 clone. The gene fragment contains entire GR gene exon Ⅱ, and two flanking fragments which are 3.9 kb and 1.4 kb respectively can be used as homologous arm in the construction of the targeting vector.