Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors.
Replacement vectors with genomic 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 allelic differences which were typed by partial sequencing. These included base pair mismatches (2%) and a polymorphic [GTC]-repeat length variation. Both in separate transfections 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.
van Deursen
J
J
Department of Cell Biology and Histology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands.
Wieringa
B
B
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics*; D004622:Embryo, Mammalian; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013234:Stem Cells
D003402:Creatine Kinase