摘要
ＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）是一种对ＥＳ（ｅｍｂｒｙｏｎｉｃｓｔｅｍ）细胞定点整合重组子进行鉴定的有效方法。由于在定点整合重组子和非定点整合重组子中外源导入基因与基因组ＤＮＡ分子整合的方式各不相同。因此，非重组子、定点整合重组子和非定点整合重组子的基因组ＤＮＡ分子结构也将各不相同。当我们设计合适的引物，从ＰＣＲ扩增结果中即可分析、鉴别出定点整合重组子、非定点整合重组子、或非重组子。本文采用ＰＣＲ方法，根据ｐＲＶ４．０质粒转化ＥＳ细胞（雄性小鼠ＣＣＥ株系）后的不同类型转化子细胞基因组ＤＮＡ分子的特点，设计了两对不同的引物，分别对转化细胞经抗Ｇ４１８，６ＴＧ初步筛选的克隆进行了分析，从中快速、简便、特异性极强地得到了定点整合重组子。我们还用ｓｏｕｔｂｅｒｎ杂交验证了这种方法的准确性。
PCR is an effective method for detecting homologous recombinants from ES cells,because there are different genomic DNA structure among non-recombinants,site specific recombinants and non-site specific recombinants.When we design suitable primers,we can analyze and identify site specific recombinants,non-site specific recombinants and non-recombinants.In this paper,we design two different pairs of primer(according to the genomic DNA patterns) for using PCR to detect the cells which resist to G418 and 6-TG after plasmid pRV4.0 transforming male mouse ES cells.we get the homologous recombinants quickly,simply and specifically.We test the reliability of PCR using southern blot hybridization.
