title: Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells.
abstract: We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10(-4), these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.
authors.affiliation: FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Pierce
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
authors.forename: O
authors.initials: O
authors.lastname: Igoucheva
authors.forename: R
authors.initials: R
authors.lastname: Omarrudin
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.forename: S L
authors.initials: SL
authors.lastname: Diamond
authors.forename: K
authors.initials: K
authors.lastname: Yoon
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D016466:CHO Cells; D006224:Cricetinae; D004277:DNA, Single-Stranded / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase