Investigation of ectopic changes between healthy and Ataxia Telangiectasia cells Open Access
Seong, Rachel (Spring 2024)
Abstract
Ataxia Telangiectasia (AT) affects 1 in 40,000 to 100,000 people. It is an autosomal recessive neurodegenerative disease that affects the part of the brain that controls motor movement. Mutations in the ataxia telangiectasia mutated (ATM) gene cause the disease, so we wanted to explore the differences in various parts of the DNA damage response pathway, such as γH2A.X, 53BP1, R-loops, and ATM, between healthy cells and AT patient-derived cells, more specifically in neural progenitor cells from age-matched healthy controls and AT patients. To investigate the differences, ionizing radiation was done to induce DNA damage in both the control and AT cell lines and given different recovery time points, which was followed by immunofluorescence staining on γH2A.X and 53BP1, as well as western blots to quantify the levels of γH2A.X. We discovered that the increase in γH2A.X levels was not as significant in AT cells when compared to the controls. Additionally, dot blots were used to assess global R-loop changes in AT cells, in which R-loop accumulation was higher in AT cells, with a further increase in R-loop accumulation after IR treatment. qPCR sequencing was done to compare ATM mRNA expression, and we discovered that there were lower levels of ATM mRNA expression in AT cells. From the data, we can conclude that AT is potentially caused by dysregulation of the DDR pathway, specifically through impairment or less transcription of ATM.
Table of Contents
Abstract
Introduction
Methods and Materials
Cell-Line Information
Reprogramming LCLs and Fibroblasts
Ionizing Radiation (IR)
RT-qPCR
Immunofluorescence Staining
Western Blots
gDNA Isolation
Dot Blots
Results
AT Cells Present Lower Expression of ATM mRNA
Differences in γH2A.X and 53BP1 Levels Between Healthy and AT Cells
R-loop Accumulation in AT
Discussion
AT Cells Present Lower Expression of ATM mRNA
Differences in γH2A.X and 53BP1 Levels Between Healthy and AT Cells
R-loop Accumulation in AT
References
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Investigation of ectopic changes between healthy and Ataxia Telangiectasia cells () | 2024-04-09 13:54:59 -0400 |
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