Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease

Mol Ther. 2010 Jun;18(6):1103-10. doi: 10.1038/mt.2010.57. Epub 2010 Apr 13.


Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA Primers
  • Disease Models, Animal*
  • Embryonic Stem Cells / metabolism
  • Gene Targeting
  • Genes, Recessive*
  • Genetic Diseases, Inborn / genetics
  • Genetic Diseases, Inborn / therapy*
  • Green Fluorescent Proteins / genetics
  • Immunocompetence
  • Mice
  • Recombination, Genetic*
  • Targeted Gene Repair*
  • Zinc Fingers*


  • DNA Primers
  • Green Fluorescent Proteins