Dissertations and Theses

Date of Award

2019

Document Type

Thesis

Department

Biology

First Advisor

Bao Q. Vuong

Second Advisor

Karen Hubbard

Third Advisor

Shireen Saleque

Keywords

Class switch recombination, metaphase spreads, B-cells, MEFs, DNA repair, genomic instability

Abstract

Ataxia telangiectasia mutated (ATM) and mutS homologue 2 (MSH2) are important DNA repair proteins that participate in DNA repair pathways to maintain genomic integrity. Mice deficient for ATM and MSH2 mice are viable. However, ATM-/- mice show growth retardation, neurological defects, and spontaneous lymphomagenesis. MSH2-/- mice suffer from aggressive lymphoid tumors between two to five months of age and have increased microsatellite instability, which predisposes MSH2-/- mice to carcinomas. However, mice deficient in both ATM and MSH2 are unable to survive beyond postnatal day 21 (P21). The observed lethality in ATM-/-MSH2-/- mice may result from the accumulation of genomic instability. To address this hypothesis, metaphase spreads were examined for chromosomal aberrations in B cells, which undergo genetically programmed DNA recombination and mutation. To bypass the lethality in ATM-/-MSH2-/- mice, a conditional knockout of ATM was bred onto an MSH2-/- background strain to generate CD21cre+ATMF/FMSH2-/- mice, which are referred to as ATM∆/∆MSH2-/-. In these mice, Cre expression, which is regulated by the CD21 promoter, deletes floxed ATM alleles in naïve mature MSH2-/- B-cells. ATM∆/∆ B-cells showed low levels of chromosomal aberrations whereas ATM∆/∆MSH2-/-B-cells had levels of chromosomal aberrations that was comparable to MSH2-/- B-cells. The latter result suggested that ATM was incompletely deleted in ATM∆/∆ B-cells. Analysis of ATM∆/∆ B cell lysates showed residual ATM protein that may account for the comparable chromosomal aberrations in ATM∆/∆MSH2-/-and MSH2-/- metaphase spreads. ATM-/-MSH2-/- embryos die in utero at approximately E16-E20. Thus, mouse embryonic fibroblasts (MEFs) were isolated from E13.5-15.5 ATM-/-MSH2-/- embryos and examined for chromosomal aberrations. The ATM-/-MSH2-/- MEF metaphase spreads displayed a similar pattern of chromosomal aberrations as ATM-/- MEFs, which does not support the hypothesis that ATM-/-MSH2-/- mice die from increased genomic instability; however, additional metaphase analyses are required to conclusively determine whether genomic instability does or does not impair ATM-/-MSH2-/- embryonic development.

Available for download on Wednesday, June 05, 2024

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