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PATHWAYS TO MUTATION IN SOMATIC AND STEM CELLS

Cervantes, Rachel Bolante
http://rave.ohiolink.edu/etdc/view?acc_num=ucin976113478

Abstract Details

2000, PhD, University of Cincinnati, Medicine : Cell and Molecular Biology.
Embryonic stem (ES) cells are intrinsically different from somatic cells in that they retain the capacity to differentiate into multiple cell types (Nagy et al., 1993; Brook and Gardner, 1997). Damage to any cell can have serious consequences, but the consequences of mutation in early embryonic cells are increased as these cells will eventually give rise to numerous progeny. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differed from that in somatic cells. The mutation rate in ES cells was significantly lower than in mouse embryonic fibroblasts (MEFs) and the distribution of mutagenic events was remarkably different between the two cell types. Loss of the functional allele was the predominant mutation type in both cases, representing about 80% of all events. However, mitotic recombination accounted for all LOH events detected in somatic cells whereas chromosome loss/reduplication, leading to uniparental disomy (UPD) was the predominant mechanism of mutation in ES cells. Accumulation of UPD with continued culture will lead to reduction to homozygosity at multiple recessive disease loci and may limit the potential clinical use of ES cells. In addition to its usefulness as a reporter of mutation in vitro, Aprt can be used to detect mutation in situ. The ability to visually distinguish between APRT proficient and deficient cells forms the basis of an APRT mouse model to detect point mutations in vivo. The overall strategy is to create mice that are Aprt-/- and whose mutant alleles contain known inactivating point mutations that are revertible when exposed to a mutagenic compound or the environment. Radiolabeled adenine that is injected intraperitoneally is only incorporated into cells that have reverted the point mutation and subsequently gained APRT activity. To establish the sensitivity of the APRT mutagenesis model, the APRT null (Aprtneo/neo) mouse was used as a syngeneic mouse model for detecting transplanted APRT wild type donor hematopoietic cells. Rare APRT+ donor cells were easily detected in the blood and tissues of recipient mice by PCR and autoradiography. Interestingly, the pattern of engraftment of primitive hematopoietic cells differed between nonirradiated and sublethally irradiated mice. The findings indicate that the Aprt-/- mouse mutagenesis model currently being produced will provide a sensitivie and effective method of detecting mutation in situ.
Peter Stambrook (Advisor)
ES cell; mutation; genomic instability; loss of heterozygosity; aprt

Recommended Citations

Citations

  • Cervantes, R. B. (2000). PATHWAYS TO MUTATION IN SOMATIC AND STEM CELLS [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin976113478

    APA Style (7th edition)

  • Cervantes, Rachel. PATHWAYS TO MUTATION IN SOMATIC AND STEM CELLS. 2000. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin976113478.

    MLA Style (8th edition)

  • Cervantes, Rachel. "PATHWAYS TO MUTATION IN SOMATIC AND STEM CELLS." Doctoral dissertation, University of Cincinnati, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=ucin976113478

    Chicago Manual of Style (17th edition)

ucin976113478
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This open access ETD is published by University of Cincinnati and OhioLINK.