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 (SFH R), has been used to correct specific genomic lesions in human epithelial c ells. 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. A s an alternative to targeting CFTR, targeted correction of a mutant selecta ble marker or reporter gene would be more amenable to accurate and rapid qu antification of gene targeting efficiency. The present study evaluates the conditions that modulate SFHR-mediated correction of a defective Zeocin ant ibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insert ion. The conditions include delivery systems, plasmid-to-fragment ratio, fr agment length, and fragment strandedness (single- or double-stranded DNA). Targeting fragments comprise the wild-type Zeo(r) gene sequence and were ei ther 410 (Zeo1) or 458 bp (Zeo3). Expression vectors containing the correct ed Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells. Correction of the Z eo(r) gene was phenotypically defined as restoration of resistance to Zeoci n in either bacteria or epithelial cell clones. Extrachromosomal gene corre ction was assayed using polymerase chain reaction amplification, restrictio n enzyme digestion, DNA sequencing, and Southern blot hybridization analysi s of DNA from isolated prokaryotic and eukaryotic clones. Neither random se quence 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 m odulated for the optimization of SFHR-mediated targeting.