Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments.
A novel gene targeting strategy, small fragment homologous replacement (SFHR), has been used to correct specific genomic lesions in human epithelial cells. The frequency of targeting was estimated to be 1-10%. However, given the genomic target, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, it is difficult to accurately quantify targeting frequency. As an alternative to targeting CFTR, targeted correction of a mutant selectable marker or reporter gene would be more amenable to accurate and rapid quantification of gene targeting efficiency. The present study evaluates the conditions that modulate SFHR-mediated correction of a defective Zeocin antibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insertion. The conditions include delivery systems, plasmid-to-fragment ratio, fragment length, and fragment strandedness (single- or double-stranded DNA). Targeting fragments comprise the wild-type Zeo(r) gene sequence and were either 410 (Zeo1) or 458 bp (Zeo3). Expression vectors containing the corrected Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells. Correction of the Zeo(r) gene was phenotypically defined as restoration of resistance to Zeocin in either bacteria or epithelial cell clones. Extrachromosomal gene correction was assayed using polymerase chain reaction amplification, restriction enzyme digestion, DNA sequencing, and Southern blot hybridization analysis of DNA from isolated prokaryotic and eukaryotic clones. Neither random sequence alteration in the target episomal gene nor random integration of the small fragments was detected. Targeted correction efficiencies of up to 4% were attained. These studies provide insight into parameters that can be modulated for the optimization of SFHR-mediated targeting.
Human Molecular Genetics Unit, University of Vermont, Colchester, Vermont 05446, USA.
A
A
Colosimo
K K
KK
Goncz
G
G
Novelli
B
B
Dallapiccola
D C
DC
Gruenert
D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D015139:Blotting, Southern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004278:DNA, Superhelical; D004352:Drug Resistance, Microbial / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection
C505032:CFTR protein, human; D004278:DNA, Superhelical; D001761:Bleomycin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C105427:Zeocin; D004247:DNA; D004262:DNA Restriction Enzymes