Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells.
A unique hybrid oligonucleotide composed of both RNA and DNA has been shown to correct a point mutation in a site-specific and inheritable manner in extrachromosomal and chromosomal targets. In order to develop new gene therapeutics for skin, we tested two oligonucleotides that were shown to create a point mutation in alkaline phosphatase and beta-globin genes in several epithelial cell types. Highly transformed epithelial cells (HeLa) exhibited a conversion frequency of 5% by both RNA-DNA oligonucleotides. In comparison, other immortalized epithelial cells (HaCaT) or human primary keratinocytes did not show any detectable level of gene conversion by the restriction fragment length polymorphism analysis, indicating less than 1% conversion frequency. The concentration of the oligonucleotide in the nuclei of HeLa cells was similar to that of HaCaT or human primary keratinocytes measured by a radiolabeled or a fluorescein-conjugated oligonucleotide. Moreover, the RNA-DNA oligonucleotide exhibited a prolonged stability in the nucleus. Thus, neither uptake nor nuclear stability of the oligonucleotide appears to be a limiting factor in gene targeting events under our experimental conditions. These results indicate that the frequency of gene targeting varies among different cells, suggesting that cellular recombination and DNA repair activities may be important.
Santana
E
E
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Peritz
A E
AE
Iyer
S
S
Uitto
J
J
Yoon
K
K
D000469:Alkaline Phosphatase / Q000235:genetics; D015816:Cell Adhesion Molecules, Neuronal / Q000494:pharmacology; D002460:Cell Line; D058969:Contactin 2; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D018390:Gene Targeting; D005914:Globins / Q000235:genetics; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology; D008297:Male; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000235:genetics*; D010761:Phosphorus Radioisotopes; D017354:Point Mutation; D012313:RNA / Q000235:genetics*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000494:pharmacology
C548674:CNTN2 protein, human; D015816:Cell Adhesion Molecules, Neuronal; D058969:Contactin 2; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D012313:RNA; D005914:Globins; D004247:DNA; D000469:Alkaline Phosphatase