Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting

The creation of precise clinical mutations by gene targeting is important i n elucidating disease pathogenesis using mouse models. 'Hit and run' gene t argeting is an elegant method to achieve this goal. This uses first a posit ive selection to introduce the targeting vector carrying the required mutat ion and then a negative selection to identify clones which have removed vec tor and wild-type sequences by intrachromosomal recombination. However, thi s approach has only been successfully used in a handful of cases. We used t his procedure to introduce precise clinical mutations into the exon 10 regi on of the cystic fibrosis transmembrane conductance regulator(Cftr) gene. U sing a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative se lection step. This defect in thymidine kinase induced toxicity to gancyclov ir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunate ly these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk select ion cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with n o false positives and successful germline transmission of the novel G480C m issense mutation.