Transgenic Research

, Volume 9, Issue 1, pp 55–66

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

  • Paul Dickinson
  • Wendy L. Kimber
  • Fiona M. Kilanowski
  • Sheila Webb
  • Barbara J. Stevenson
  • David J. Porteous
  • Julia R. Dorin
Article

DOI: 10.1023/A:1008915026660

Cite this article as:
Dickinson, P., Kimber, W.L., Kilanowski, F.M. et al. Transgenic Res (2000) 9: 55. doi:10.1023/A:1008915026660

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 selectable 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.

cystic fibrosis ES cells gene inactivation gene targeting hit and run 

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • 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. 1.MRC Human Genetics Unit, Western General HospitalEdinburgh
  2. 2.Geron Bio-Med, Roslin InstituteRoslin
  3. 3.Department of PhysiologyJohns Hopkins University, School of MedicineBaltimore
  4. 4.Medical Genetics Section, Department of Medical SciencesUniversity of Edinburgh, Molecular Medicine Centre, Western General HospitalEdinburgh

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