A novel selectable system for detecting expansion of CAG.CTG repeats in mammalian cells.

Details

Serval ID
serval:BIB_B8909E830008
Type
Article: article from journal or magazin.
Collection
Publications
Title
A novel selectable system for detecting expansion of CAG.CTG repeats in mammalian cells.
Journal
Mutation Research
Author(s)
Lin Y., Dion V., Wilson J.H.
ISSN
0027-5107 (Print)
ISSN-L
0027-5107
Publication state
Published
Issued date
2005
Volume
572
Number
1-2
Pages
123-131
Language
english
Abstract
CAG.CTG repeat expansions cause more than a dozen neurodegenerative diseases in humans. To define the mechanism of repeat instability in mammalian cells we developed a selectable assay to detect expansions of CAG.CTG triplet repeats in Chinese hamster ovary (CHO) cells. We showed previously that long tracts of CAG.CTG repeats, embedded in an intron of the APRT gene, kill expression of the gene, rendering the cells APRT-. By contrast, tracts with fewer than 34 repeats allow sufficient expression to give APRT+ cells. Although it should be possible to use APRT+ cells with short repeats to assay for expansion events by selecting for APRT- cells, we find that APRT+ cells with 31 repeats are not killed by the standard APRT- selection protocol, most likely because they produce too little Aprt to incorporate sufficient 8-azaadenine into their adenine pool. To overcome this problem, we devised a new selection, which increases the proportion of the adenine pool contributed by the salvage pathway by partially inhibiting the de novo pathway. We show that APRT- CHO cells with 61 or 95 CAG.CTG repeats survive this selection, whereas cells with 31 repeats die. Using this selection system, we can select for expansion to as few as 39 repeats. Thus, this assay can monitor expansions across the critical boundary from the longest lengths of normal alleles to the shortest lengths of disease alleles.
Keywords
Adenine Phosphoribosyltransferase/genetics, Animals, Base Sequence, CHO Cells, Cricetinae, DNA Primers, Polymerase Chain Reaction, Trinucleotide Repeats
Pubmed
Web of science
Create date
27/02/2014 9:58
Last modification date
20/08/2019 15:26
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