Ratcliff R, Evans M J, Doran J, Wainwright B J, Williamson R, Colledge W H
Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, UK.
Transgenic Res. 1992 Jul;1(4):177-81. doi: 10.1007/BF02522536.
We have successfully disrupted the cftr (cystic fibrosis transmembrane conductance regulator) gene at its endogenous locus in embryonic stem cells by gene targeting. We are using a double replacement strategy to introduce subtle mutations into exon 10. We report here the first step of creating a null mutation by insertion of a functional hprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of the cftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of the cftr targeted clones has been injected into recipient blastocysts and shown to contribute to chimaeras. The targeted clones will now be used as the starting point for a second gene targeting step to remove the hprt gene in exon 10 with the concomitant introduction of the delta F508 mutation or other mutations.
我们已通过基因打靶成功地在胚胎干细胞的内源性位点破坏了cftr（囊性纤维化跨膜传导调节因子）基因。我们正在使用双替换策略将微小突变引入外显子10。我们在此报告通过将功能性hprt（次黄嘌呤磷酸核糖基转移酶）小基因插入cftr基因的外显子10来创建无效突变的第一步。通过PCR筛选鉴定靶向胚胎干细胞克隆，并通过Southern印迹分析进行确认。其中一个cftr靶向克隆已注射到受体囊胚中，并显示对嵌合体有贡献。现在，靶向克隆将用作第二步基因打靶的起点，以去除外显子10中的hprt基因，同时引入ΔF508突变或其他突变。
