Transgenic Research 9: 55–66, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 55 Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting Paul Dickinson 1,2,† , Wendy L. Kimber 1,3,† , Fiona M. Kilanowski 1 , Sheila Webb 1 , Barbara J. Stevenson 1,4 , David J. Porteous 1,4 & Julia R. Dorin 1, * 1 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU 2 Geron Bio-Med, Roslin Institute, Roslin, Midlothian, EH25 9PS 3 Department of Physiology, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185 4 Medical Genetics Section, Department of Medical Sciences, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh EH4 2XU Received 4 November 1999; revised 18 January 2000; accepted 19 January 2000 Key words: cystic fibrosis, ES cells, gene inactivation, gene targeting, hit and run Abstract The creation of precise clinical mutations by gene targeting is important in elucidating disease pathogenesis using mouse models. ‘Hit and run’ gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative se- lectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true ‘run’ clones. Unfortunately 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 selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient ‘hit and run’ for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation. Abbreviations: CFTR – cystic fibrosis transmembrane conductance regulator; CMV – Cytomeglavirus; HSV – Herpes Simplex virus; hyg – hygromycin; hyg/tk – hygromycin/thymidine kinase fusion gene; neo – neomycin; tk – thymidine kinase Introduction Gene knockouts in the mouse can provide good mod- els for human disease but the investigation of gen- otype/phenotype correlation require the introduction of precise mutations to the locus of interest. Gene * Author for correspondence Tel.: +44 (0)131 467 8415; Fax: +44 (0)131 343 2620; E-mail: julia.dorin@hgu.mrc.ac.uk † Paul Dickinson and Wendy L. Kimber contributed equally to the study. targeting to successfully carry out this analysis has been performed using a range of techniques (Deng et al., 1993; Hasty et al., 1991; Thomas et al., 1987), which vary greatly in the subtlety with which an alteration may be introduced. At one end of the range, introduction of selectable markers in an in- tron along with a precise mutation may significantly modify expression of the targeted locus (Colledge et al., 1995; Delaney et al., 1996; Fiering et al., 1995).