Tsuda H, Maynard-Currie C E, Reid L H, Yoshida T, Edamura K, Maeda N, Smithies O, Jakobovits A
Cell Genesys, Inc., Foster City, California 94404, USA.
Genomics. 1997 Jun 15;42(3):413-21. doi: 10.1006/geno.1997.4771.
To obtain useful hypoxanthine phosphoribosyl-transferase (HPRT)-deficient mouse ES cell lines, two different methods were employed: (i) selection of spontaneous 6-TG-resistant mutants and (ii) gene targeting of the HPRT locus. The first approach resulted in the establishment of E14.1TG3B1, a spontaneous HPRT-deficient cell line with an insertional mutation of 203 bp in the third exon of the HPRT gene. The insert is highly homologous to the B2 mouse repetitive element and has all the expected retroposon characteristics, thus providing an example of gene inactivation by retroposon insertion. This clone exhibited stable 6-TG resistance and high germ-line transmission frequency. Thus E14.1TG3B1 is a useful ES cell line for modifying the mouse genome using the HPRT gene as a selection marker and for transmission at a high frequency into the mouse germ line. The second approach resulted in a 55-kb deletion of the mouse HPRT locus, demonstrating the feasibility of replacement-targeting vectors to generate large genomic DNA deletions.
为获得有用的次黄嘌呤磷酸核糖转移酶（HPRT）缺陷型小鼠胚胎干细胞系，采用了两种不同方法：（i）筛选自发的6-硫鸟嘌呤（6-TG）抗性突变体，以及（ii）对HPRT基因座进行基因打靶。第一种方法产生了E14.1TG3B1，这是一种自发的HPRT缺陷型细胞系，在HPRT基因的第三个外显子中有一个203 bp的插入突变。该插入片段与B2小鼠重复元件高度同源，并具有所有预期的反转录转座子特征，从而提供了一个通过反转录转座子插入导致基因失活的例子。该克隆表现出稳定的6-TG抗性和高种系传递频率。因此，E14.1TG3B1是一种有用的胚胎干细胞系，可用于以HPRT基因为选择标记修饰小鼠基因组，并以高频率传递到小鼠种系中。第二种方法导致小鼠HPRT基因座出现55 kb的缺失，证明了置换打靶载体产生大片段基因组DNA缺失的可行性。
