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Comparative analysis of protocols used in testing effects of high oxygen culture conditions on the...

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Comparative analysis of protocols used in testing effects of high oxygen culture conditions on the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content in differentially methylated regions (DMR) of imprinted genes in bovine in vitro fertilized (IVF) blastocysts

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Identifier	Thesis 2486
Author	Duong, Linh (Linh Mong), 1981-
Title	Comparative analysis of protocols used in testing effects of high oxygen culture conditions on the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content in differentially methylated regions (DMR) of imprinted genes in bovine in vitro fertilized (IVF) blastocysts
Publisher	Central Connecticut State University
Date of Publication	2015
Resource Type	Master's Thesis
Abstract	While it was only a few years ago that human in vitro fertilization (IVF) was routinely conducted under high atmospheric oxygen conditions (~20% oxygen) -- a stark contrast to estimated 5% oxygen levels found in vivo -- studies have demonstrated that differing oxygen concentrations can significantly impact embryonic development. We hypothesized that embryos cultured under 20% oxygen will have increased levels of TET1-3 expression leading to greater conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) ultimately resulting in DNA demethylation. Imprinting disorders are often caused by the loss of imprinting due to DNA demethylation and the biallelic expression of imprinted genes. The goal of this study was to develop and optimize protocol conditions to assess the effects 5% and 20% oxygen on 5mC and 5hmC content in differentially methylated regions (DMR) of imprinted genes in bovine IVF blastocysts. DNA was extracted from commercially generated bovine IVF blastocysts, fragmented by sonication, and enriched for 5mC and 5hmC. Next, the DNA was purified and amplified using a whole genome amplification kit. Lastly, real time quantitative polymerase chain reaction (qPCR) was used to quantify the 5mC and 5hmC-enriched DNA. My study identified key steps in several protocols that were important in assessing methylation patterns: 1) the selection of the buffer for sonication was essential to ensure fragmented DNA was between 200–500 base pairs for enrichment; 2) proper primer design was fundamental to assessing differences in enrichment; and 3) most importantly, the method used for DNA extraction was a key determining factor in the success of downstream experiments as low DNA yield reduced the effectiveness of enrichment and subsequent amplification. This thesis is an important step in constructing objective tools to evaluate the effects of the culture environment on methylation patterns in a small numbers of embryos.
Notes	"Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology, Health Sciences Specialization, Department of Biology."; Thesis advisor: Sadie Marjani.; M.S.,Central Connecticut State University,,2015.; Includes bibliographical references (leaves 61-68).
Subject	Epigenetics. Genomic imprinting.
Department	Department of Biological Sciences
Advisor	Marjani, Sadie
Type	Text
Digital Format	application/pdf
Software	System requirements: PC and World Wide Web browser.
Language	eng
OCLC number	940805984
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Comparative analysis of protocols used in testing effects of high oxygen culture conditions on the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content in differentially methylated regions (DMR) of imprinted genes in bovine in vitro fertilized (IVF) blastocysts

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