Gene Targeting by Oligonucleotides in Keratinocytes View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2004-10-01

AUTHORS

Kursad Turksen , Olga Igoucheva , Kyonggeun Yoon

ABSTRACT

Oligonucleotide-directed gene alteration produces a targeted deoxyribonucleic acid (DNA) sequence change in the genome of mammalian cells at low frequency that is only detectable by highly sensitive methods. To measure the low frequency, we have established an assay using the mutant lacZ vector that contains a single point mutation in the lacZ gene, which results in a loss of enzymatic activity. When cells containing this mutant reporter gene are corrected by gene targeting, the mutant β-galactosidase enzymatic activity is restored, and corrected cells can be visualized by histochemical staining. Using this method, we detected a low level of gene correction in the primary human keratinocytes, in spite of highly efficient nuclear uptake of oligonucleotide. Therefore, it is important to consider many other factors for successful gene repair, including DNA repair and recombination activities, status of replication and transcription, in addition to the well-known requirements like the quality and delivery of oligodeoxynucleotides to cells. Available methods to manipulate epidermal stem cells and the accessibility of the tissue make the epidermis attractive for gene targeting. Given the low frequency, however, general selection procedures and amplification of corrected cells via epidermal stem cells are ultimately needed to make the gene repair technology practical. More... »

PAGES

287-302

Book

TITLE

Epidermal Cells

ISBN

1-59259-830-7

Author Affiliations

Identifiers

URI

http://scigraph.springernature.com/pub.10.1385/1-59259-830-7:287

DOI

http://dx.doi.org/10.1385/1-59259-830-7:287

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1044615292


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