title: Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease.
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.
authors.affiliation: Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Jon P
authors.initials: JP
authors.lastname: Connelly
authors.forename: Jenny C
authors.initials: JC
authors.lastname: Barker
authors.forename: Shondra
authors.initials: S
authors.lastname: Pruett-Miller
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000378:metabolism; D018390:Gene Targeting; D005808:Genes, Recessive; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000628:therapy*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007121:Immunocompetence; D051379:Mice; D011995:Recombination, Genetic; D052416:Targeted Gene Repair; D016335:Zinc Fingers
chemical_list: D017931:DNA Primers; D049452:Green Fluorescent Proteins
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