NDLI: Identifying DNA mutations in purified hematopoietic stem/progenitor cells.

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Year: 2014 Issue: 83

Identifying DNA mutations in purified hematopoietic stem/progenitor cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Cheng, Ziming Zhou, Ting Merchant, Azhar Prihoda, Thomas J. Wickes, Brian L. Xu, Guogang Walter, Christi A. Rebel, Vivienne I.
Description	Author Affiliation: Cheng Z ( Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio.); Zhou T ( Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio); Merchant A ( Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio.); Prihoda TJ ( Department of Pathology, UT Health Science Center at San Antonio.); Wickes BL ( Department of Microbiology, UT Health Science Center at San Antonio); Xu G ( Department of Cellular and Structural Biology, UT Health Science Center at San Antonio.); Walter CA ( Department of Cellular and Structural Biology, UT Health Science Center at San Antonio); Rebel VI ( Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio)
Abstract	In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases. LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of ß-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation. The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin(-)IL7R(-)Sca-1(+)cKit(++)(LSK) cells and other subpopulations of the hematopoietic system.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/50752
Journal	Journal of Visualized Experiments
Issue Number	84
Language	English
Publisher	MyJove Corp.
Publisher Date	2014-02-24
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Dna Mutational Analysis Hematopoietic Stem Cells Physiology Animals Bacteriophage Lambda Genetics Genetic Vectors Chemistry Lac Repressors Mice Mice, Inbred C57bl Mice, Transgenic Mutagenesis Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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