title: Sequence-specific correction of genomic hypoxanthine-guanine phosphoribosyl transferase mutations in lymphoblasts by small fragment homologous replacement.
abstract: Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specific modification of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modification strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the efficiency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fluorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.
authors.affiliation: California Pacific Medical Center Research Institute, San Francisco, California 94107, USA.
authors.forename: Babak
authors.initials: B
authors.lastname: Bedayat
authors.forename: Alireza
authors.initials: A
authors.lastname: Abdolmohamadi
authors.forename: Lin
authors.initials: L
authors.lastname: Ye
authors.forename: Rosalie
authors.initials: R
authors.lastname: Maurisse
authors.forename: Hooman
authors.initials: H
authors.lastname: Parsi
authors.forename: Jennifer
authors.initials: J
authors.lastname: Schwarz
authors.forename: Hamid
authors.initials: H
authors.lastname: Emamekhoo
authors.forename: Janice A
authors.initials: JA
authors.lastname: Nicklas
authors.forename: J Patrick
authors.initials: JP
authors.lastname: O'Neill
authors.forename: Dieter C
authors.initials: DC
authors.lastname: Gruenert
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D003470:Culture Media; D017931:DNA Primers; D023281:Genomics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008214:Lymphocytes / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase