Nucleic Acids Research, 1992, Vol. 20, No. 15 3815-3820
© 1992
MOLECULAR BIOLOGY |
Targeting of the creating kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors
Department of Cell biology and histology, Faculty of Medicine Sciences, University of Nijmegen PO Box 9101, 6500 HB Nijmgen, The Netherlands
*To whom correspondence should be addressed
Received June 15, 1992. Accepted July 9, 1992.
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.
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