title: Identifying DNA mutations in purified hematopoietic stem/progenitor cells.
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 β-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.
authors.affiliation: Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio.
authors.forename: Ziming
authors.initials: Z
authors.lastname: Cheng
authors.affiliation: Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio; Department of Cellular and Structural Biology, UT Health Science Center at San Antonio.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio.
authors.forename: Azhar
authors.initials: A
authors.lastname: Merchant
authors.affiliation: Department of Pathology, UT Health Science Center at San Antonio.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Prihoda
authors.affiliation: Department of Microbiology, UT Health Science Center at San Antonio; Cancer Therapy and Research Center, UT Health Science Center at San Antonio.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Wickes
authors.affiliation: Department of Cellular and Structural Biology, UT Health Science Center at San Antonio.
authors.forename: Guogang
authors.initials: G
authors.lastname: Xu
authors.affiliation: Department of Cellular and Structural Biology, UT Health Science Center at San Antonio; Cancer Therapy and Research Center, UT Health Science Center at San Antonio.
authors.forename: Christi A
authors.initials: CA
authors.lastname: Walter
authors.affiliation: Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio; Department of Cellular and Structural Biology, UT Health Science Center at San Antonio; Cancer Therapy and Research Center, UT Health Science Center at San Antonio; rebel@uthscsa.edu.
authors.forename: Vivienne I
authors.initials: VI
authors.lastname: Rebel
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000502:physiology*; D056968:Lac Repressors / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016296:Mutagenesis
chemical_list: D056968:Lac Repressors; D004247:DNA