title: Isolating gene-corrected stem cells without drug selection.
abstract: Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >/=10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a "purity" of >30%, enriched >2,000-fold and with a recovery of approximately 20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >/=3 days.
authors.affiliation: Departments of Pathology and Laboratory Medicine, and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
authors.forename: Seigo
authors.initials: S
authors.lastname: Hatada
authors.forename: Larry W
authors.initials: LW
authors.lastname: Arnold
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hatada
authors.forename: John E
authors.initials: JE
authors.lastname: Cowhig
authors.forename: Dominic
authors.initials: D
authors.lastname: Ciavatta
authors.forename: Oliver
authors.initials: O
authors.lastname: Smithies
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D018390:Gene Targeting; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase