Targeting of the creating kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors

Jan Van Deursen, Beè Wieringa

Research output: Contribution to journalArticle

68 Scopus citations

Abstract

Replacement vectors with genomlc DNA originating from different mouse strains were used to introduce site-specific mutations into the creatine kinase M (CKM) gene of mouse embryonic stem (ES) cells. Here we demonstrate that in mouse strain 129-derived ES cells, the gene is at least 25-fold more efficiently targeted with an Isogenic, 129-derived vector (129-pRV8.3) than with a nonisogenic, BALB/c-specific vector (BALB/c-pRV8.3). The two targeting constructs were identical except for allellc differences which were typed by partial sequencing. These included base pair mismatches (2%) and a polymorphic [GTC]-repeat length variation. Both in separate transfectlons as well as in cotransfections with mixed vectors, homologous disruption of the CKM gene resulted uniquely from the 129-isogenic DNA. Our data confirm earlier observations on requirements for homologous recombination in pro- and eukaryotic systems and indicate that targeting of the CKM locus is highly sensitive to small sequence differences between cognate segments in the endogenous and incoming DNA.

Original languageEnglish (US)
Pages (from-to)3815-3820
Number of pages6
JournalNucleic acids research
Volume20
Issue number15
DOIs
StatePublished - Aug 11 1992

ASJC Scopus subject areas

  • Genetics

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