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

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

Author information

  • 1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Abstract

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.

PMID:
20389291
[PubMed - indexed for MEDLINE]
PMCID:
PMC2889743
Free PMC Article

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