Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes.
Various oligonucleotide (ODN)-based approaches have been proposed for their ability to correct mutated genes at the normal chromosomal locations. However, the reported gene correction frequencies of these approaches have varied markedly in different experimental settings, including when different tissues or cell types are targeted. In order to find the optimal ODN-based approach for a specific target tissue, an assay system that allows direct comparison of the different methods on that tissue is necessary. Herein, we describe an XP-UVC selection assay that can be used to evaluate and compare gene correction frequencies in different cell types obtained from a xeroderma pigmentosum (XP) patient, following treatment by different ODN-based approaches. As an experimental example, the XP-UVC selection assay was used to assess the ability of chimeric RNA/DNA ODN to correct point mutations in the XPA gene. This assay can be used to assess and evaluate other types of ODN-based approaches, and to further optimize them.
Terunuma
A
A
Dermatology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Ye
J
J
Emmert
S
S
Khan
S G
SG
Kraemer
K H
KH
Vogel
J C
JC
D001483:Base Sequence; D016923:Cell Death / Q000528:radiation effects; D002460:Cell Line; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008214:Lymphocytes / Q000473:pathology / Q000528:radiation effects; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein