title: A comparison of gene repair strategies in cell culture using a lacZ reporter system.
abstract: Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.
authors.affiliation: Department of Physiology, University of Cambridge, Downing Street, Cambridge, UK.
authors.forename: H D
authors.initials: HD
authors.lastname: Nickerson
authors.forename: W H
authors.initials: WH
authors.lastname: Colledge
mesh_terms: D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western; D002460:Cell Line; D002875:Chromosomes; D004260:DNA Repair; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007763:Lac Operon; D011233:Precipitin Tests; D014162:Transfection / Q000379:methods*