Abstract
Assays for in vivo mutation are used to identify genotoxic hazards and phenotypes prone to genomic instability and cancer. The hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the phosphatidyl inositol glycan, class A (Pig-a) gene are endogenous X-linked genes that can be used as reporters of mutation in peripheral blood lymphocytes from most mammals. Here we describe methodology for measuring Hprt and Pig-a mutation in rat T-lymphocytes. The identification and selective expansion of mutant lymphocytes are based upon the phenotypic properties of Hprt- and Pig-a-deficient cells, i.e., resistance to the purine analog, 6-thioguanine, or to the bacterial toxin, proaerolysin. Expanded mutants can be further analyzed by sequencing cDNA from the target transcripts for identification of small sequence alterations and by multiplex PCR analysis of genomic DNA for the detection of deletions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bertram JS (2000) The molecular biology of cancer. Mol Aspects Med 21:167–223
Adair GM, Carver JH, Wandres DL (1980) Mutagenicity testing in mammalian cells. I. Derivation of a Chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci. Mutat Res 72:187–205
Lambert IB, Singer TM, Boucher SE et al (2005) Detailed review of transgenic rodent mutation assays. Mutat Res 590:1–280
Kinoshita T, Fujita M, Maeda Y (2008) Biosynthesis, remodelling and functions of mammalian GPI–anchored proteins: recent progress. J Biochem 144:287–294
Brodsky RA, Mukhina GL, Nelson KL et al (1999) Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol–binding toxin aerolysin. Blood 93:1749–1756
Dobrovolsky VN, Shaddock JG, Heflich RH (2000) 7,12–dimethylbenz[a]anthracene–induced mutation in the Tk gene of Tk+/– mice: automated scoring of lymphocyte clones using a fluorescent viability indicator. Environ Mol Mutagen 36:283–291
Miura D, Dobrovolsky VN, Mittelstaedt RA et al (2008) Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants. Environ Mol Mutagen 49:622–630
Chen T, Aidoo A, Mittelstaedt RA et al (1999) Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa. Carcinogenesis 20:269–277
Dobrovolsky VN, Shaddock JG, Heflich RH (2005) Analysis of in vivo mutation in the Hprt and Tk genes of mouse lymphocytes. In: Keohavong P, Grant SG (eds) Molecular Toxicology Protocols. Humana, Totowa, NJ, pp 133–144
Disclaimer
The views presented in this chapter do not necessarily reflect those of the US Food and Drug Administration.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Dobrovolsky, V.N., Shaddock, J.G., Mittelstaedt, R.A., Miura, D., Heflich, R.H. (2013). Detection of In Vivo Mutation in the Hprt and Pig-a Genes of Rat Lymphocytes. In: Dhawan, A., Bajpayee, M. (eds) Genotoxicity Assessment. Methods in Molecular Biology, vol 1044. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-529-3_4
Download citation
DOI: https://doi.org/10.1007/978-1-62703-529-3_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-528-6
Online ISBN: 978-1-62703-529-3
eBook Packages: Springer Protocols