Identifying DNA mutations in purified hematopoietic stem/progenitor cells.

Ziming Cheng, Ting Zhou, Azhar Merchant, Thomas J. Prihoda, Brian L Wickes, Guogang Xu, Christi A Walter, Vivienne I. Rebel

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number84
StatePublished - 2014

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Hematopoietic Stem Cells
Stem cells
DNA
Stem Cells
Genes
Mutation
Escherichia coli
Tissue homeostasis
Assays
Transgenic Mice
Galactosidases
Chromogenics
Chromogenic Compounds
Mutagenesis
Bacteriophages
Hematopoietic System
Substrates
Ports and harbors
Genomic Instability
Sepharose

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Cheng, Z., Zhou, T., Merchant, A., Prihoda, T. J., Wickes, B. L., Xu, G., ... Rebel, V. I. (2014). Identifying DNA mutations in purified hematopoietic stem/progenitor cells. Journal of visualized experiments : JoVE, (84).

Identifying DNA mutations in purified hematopoietic stem/progenitor cells. / Cheng, Ziming; Zhou, Ting; Merchant, Azhar; Prihoda, Thomas J.; Wickes, Brian L; Xu, Guogang; Walter, Christi A; Rebel, Vivienne I.

In: Journal of visualized experiments : JoVE, No. 84, 2014.

Research output: Contribution to journalArticle

Cheng, Ziming ; Zhou, Ting ; Merchant, Azhar ; Prihoda, Thomas J. ; Wickes, Brian L ; Xu, Guogang ; Walter, Christi A ; Rebel, Vivienne I. / Identifying DNA mutations in purified hematopoietic stem/progenitor cells. In: Journal of visualized experiments : JoVE. 2014 ; No. 84.
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