Gene targeting strategy used to generate point mutations in the gk gene. Uppermost map is a diagram of the mouse gk gene showing locations of exons 3 to 10 (indicated by open boxes). The locations of the DNA fragments used as 5' and 3' hybridization probes are shown. EI, EcoRI; EV, EcoRV. Second map from top is of the gene targeting vector carrying either the K414E mutation in exon 9 depicted by the red circle or the A456V mutation in exon 10 as indicated by the blue circle. A neomycin resistance cassette (pgk-neoR), which is flanked with two loxP sites depicted by red triangles, and a HSV-thymidine kinase cassette (pgk-HSV-TK), were used for positive and negative selection, respectively. Third map from top is of the recombinant gk allele after homologous recombination (HR) carrying a floxed pgk-neoR cassette and the respective point mutation in exon 9 or 10. Fourth map from top is of the mutant gk allele after Cre recombination.
M.F. Pino, K-A. Kim, K.D. Shelton, J. Lindner, S. Odili, C. Li, H.W. Collins, M. Shiota, F.M. Matschinsky and M.A. Magnuson, Glucokinase thermolability and hepatic regulatory protein binding are essential factors for predicting the blood glucose phenotype of missense mutations, Journal of Biological Chemistry, 2007 May 4, 282(18):13906-16.