Stability of HPRT marker gene expression at different gene-targeted loci: observing and overcoming a position effect.

Abstract:

: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 phosphoribosyltransferase ( HPRT) minigenes in HPRT-deficient embryonic stem cells. Our standard HPRTminigene, under the control of the mouse phosphoglycerate kinase-1 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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Melton DW,Ketchen AM,Selfridge J

doi

10.1093/nar/25.19.3937

subject

Has Abstract

pub_date

1997-10-01 00:00:00

pages

3937-43

issue

19

eissn

0305-1048

issn

1362-4962

pii

gka615

journal_volume

25

pub_type

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