科研之友
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摘要
For sophisticated gene targeting procedures requiring two sequential selective steps to operate efficiently it is essential that the marker genes used are not prone to position effects. The double replacement gene targeting procedure, to produce mice with subtle gene alterations, is based on the use of hypoxanthine phosphoriboxyltransferas (HPRT) minigenes in HPRT-deficient embryonic stem cells. Our standard HPRT minigene, under the control of the mouse phosphoglycerate kinase-l gene promoter, was stably expressed at five of six target loci examined. At the remaining locus, DNA ligase I (Lig1), expression of this minigene was highly unstable. A different minigene, under the control of the mouse HPRT promoter and embedded in its natural CpG-rich island, overcame this position effect and was stably expressed when targeted to the identical site in the Lig1 locus. The promoter region of the stably expressed minigene remained unmethylated, while the promoter of the unstably expressed minigene rapidly became fully methylated. The difference in the stability of HPRT minigene expression at the same target locus can be explained in the context of the different lengths of their CpG-rich promoter regions with associated transcription factors and a resulting difference in their susceptibility to DNA methylation, rather than by differences in promoter strength.
- 出版日期1997-10-1
