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Citation
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HERO ID
3015173
Reference Type
Journal Article
Title
Single-molecule PCR analysis of an unstable microsatellite for detecting mutations in sperm of mice exposed to chemical mutagens
Author(s)
Beal, MA; Rowan-Carroll, A; Canipbell, C; Williams, A; Somers, CM; Marchetti, F; Yauk, CL
Year
2015
Is Peer Reviewed?
1
Journal
Mutation Research
ISSN:
0027-5107
EISSN:
1873-135X
Publisher
ELSEVIER SCIENCE BV
Location
AMSTERDAM
Volume
775
Page Numbers
26-32
Language
English
PMID
25863182
DOI
10.1016/j.mrfmmm.2015.03.010
Web of Science Id
WOS:000354584900004
URL
https://linkinghub.elsevier.com/retrieve/pii/S0027510715000688
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Abstract
Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded simple tandem repeats (ESTRs), has been the most efficient method for studying germline mutation induction in endogenous sequences to date. However, the long length of these sequences makes mutation detection imprecise and laborious, and they have been characterized only in mice. Here, we explore the use of unstable microsatellite sequences that can be typed with high precision by capillary electrophoresis as alternative loci for detecting germline mutations. We screened 24 microsatellite loci across inbred mouse strains and identified Mm2.2.1 as the most polymorphic microsatellite locus. We then optimized SM-PCR of Mm2.2.1 to detect mutations in sperm. SM-PCR analysis of sperm from untreated B6C3F1 and Muta (TM) Mouse samples revealed mutation frequencies that are consistent with rates derived from family pedigree analysis (similar to 5 x 10(-3)). To determine whether this locus can be used to detect chemically induced germline mutations, Muta (TM) Mouse males were exposed by oral gavage to a single dose of 100 mg/kg of N-ethyl-N-nitrosourea (ENU) or to 100 mg/kg of benzo(a)pyrene (BaP) for 28 days alongside vehicle treated controls. Sperm were collected 10 weeks post-ENU exposure to sample sperm exposed as spermatogonial stem cells and 6 weeks post-BaP exposure to sample sperm that were dividing spermatogonia when the exposure was terminated. Both treatments resulted in a significant (approximately 2-fold) increase in mutation frequency in sperm compared to the control animals. The work establishes the utility of this microsatellite for studying mutation induction in the germ cells of mice. Because microsatellites are found in virtually every species, this approach holds promise for other organisms, including humans. Crown Copyright (C) 2015 Published by Elsevier B.V.
Keywords
Germline mutation; Sperm; Microsatellite; Single-molecule PCR; Mouse
Tags
IRIS
•
Ammonia, Oral - Problem Formulation
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