Development and Application of a Single Mouse Embryo DNA Methylation-Detection Assay
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Home > Medicine & Health Science textbooks > Medical specialties, branches of medicine > Gynaecology and obstetrics > Development and Application of a Single Mouse Embryo DNA Methylation-Detection Assay
Development and Application of a Single Mouse Embryo DNA Methylation-Detection Assay

Development and Application of a Single Mouse Embryo DNA Methylation-Detection Assay


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About the Book

This dissertation, "Development and Application of a Single Mouse Embryo DNA Methylation-detection Assay" by Chun-kit, Peter, Kwan, 關駿傑, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: During preimplantation embryonic development, imprinting genes are susceptible to methylation changes by artificial manipulation, which may lead to developmental abnormalities. In addition, environmental endocrine disruptors (EDs) in everyday household products are also found to perturb fertility development and cause epigenetic aberrations. While embryo supply is scarce and conventional epigenetic studies require embryos in vast amount, an assay was developed in this study to examine the methylation statuses of imprinting genes using DNA from single mouse blastocysts cultured in-vitro or exposed to EDs. Promoter CpG methylation patterns of three imprinting genes, small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 3 (Peg3), and potassium voltage-gated channel 1 overlapping transcript 1 (Kcnq1ot1), were examined from genomic DNA of a single mouse blastocyst. The genomic DNA was isolated and treated with bisulfite modification to preserve the methylation statuses. Afterwards, the DNA was subjected to whole genome amplification (WGA). Methylation-specific polymerase chain reaction (methyl-PCR) was performed with allele-specific primers; the amplicons were cloned and sequenced. CpG methylations in SNRPN, Peg3 and Kcnq1ot1 showed no statistical significant difference (P>0.05; Mann Whitney U test) in both parental alleles between a single genomic-amplified blastocyst and 20 non-amplified blastocysts, indicating no artifact was being introduced during the WGA procedure. Using the assay, it was revealed that blastocysts cultured in-vitro expressed slight but nonsignificant deviation in methylation rates to both parental alleles of SNRPN and Kcnq1ot1 except in single blastocysts, which displayed significant loss in maternal methylation on SNRPN upon culturing. On the other hand, paternal methylation profile of Peg3 appeared unaffected, suggesting resistance to methylation perturbations induced by in-vitro culturing. Despite that there was no significant difference in overall methylation rates between in-vivo or in-vitro developed blastocysts, certain CpG residues appeared to displayed significant loss of methylation (LOM) or gain of methylation (GOM) induced by in-vitro culture in all three genes being studied. Furthermore, using the developed, assay the epigenetic effects of three endocrine disruptors, simazine, propiconazole, and cadmium chloride (CdCl2) on in-vitro cultured single blastocysts were revealed. When compared to blastocysts cultured with KSOM+AA medium as controls, CdCl2-treated blastocysts displayed the most methylation aberrations in both alleles and within particular CpG residues, possibly due to its dual effect in both hypermethylation and hypomethylation across the methylome. Both simazine- and propiconazole -treated blastocysts displayed overall methylation significant defects were observed within particular CpG residues. Overall, the assay used in this study allowed the comprehensive investigation of methylome from the DNA extracted from a single blastocyst.defects resembled to those blastocysts cultured with KSOM+AA medium alone but DOI: 10.5353/th_b5194756 Subjects: DNA - Methylation


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Product Details
  • ISBN-13: 9781361340578
  • Publisher: Open Dissertation Press
  • Publisher Imprint: Open Dissertation Press
  • Height: 279 mm
  • No of Pages: 220
  • Weight: 522 gr
  • ISBN-10: 1361340576
  • Publisher Date: 26 Jan 2017
  • Binding: Paperback
  • Language: English
  • Spine Width: 12 mm
  • Width: 216 mm


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