Genomic Imprinting: Methods and Protocols / Edition 1

Genomic Imprinting: Methods and Protocols / Edition 1

by Andrew Ward
     
 

ISBN-10: 089603741X

ISBN-13: 9780896037410

Pub. Date: 10/28/2001

Publisher: Springer-Verlag New York, LLC

Andrew Ward and a team of experienced researchers have brought together a collection of optimized classic and vanguard techniques for the identification and analysis of imprinted genes. Protocols are included for identifying and cloning imprinted genes, for analyzing imprinted gene expression, for the study of DNA methylation and methylation-sensitive DNA-binding

Overview

Andrew Ward and a team of experienced researchers have brought together a collection of optimized classic and vanguard techniques for the identification and analysis of imprinted genes. Protocols are included for identifying and cloning imprinted genes, for analyzing imprinted gene expression, for the study of DNA methylation and methylation-sensitive DNA-binding proteins, and for examining chromatin structure. There are also methods for the manipulation of mouse embryos to produce monoparental embryos and embryonic stem cells, and for the generation of transgenic mice with BAC, PAC,and YAC constructs. Each technique is described in step-by-step detail to ensure successful results.

Product Details

ISBN-13:
9780896037410
Publisher:
Springer-Verlag New York, LLC
Publication date:
10/28/2001
Series:
Methods in Molecular Biology Series, #181
Edition description:
2002
Pages:
377
Product dimensions:
5.98(w) x 9.02(h) x 0.03(d)

Table of Contents

Prefacev
Contributorsix
1Generation of Monoparental Embryos for Investigation into Genomic Imprinting1
2Deriving and Propagating Mouse Embryonic Stem Cell Lines for Studying Genomic Imprinting21
3Balanced Translocations for the Analysis of Imprinted Regions of the Mouse Genome41
4Production of YAC Transgenic Mice by Pronuclear Injection55
5A Transgenic Approach to Studying Imprinted Genes: Modified BACs and PACs67
6Methylation-Sensitive Genome Scanning83
7Subtraction-Hybridization Method for the Identification of Imprinted Genes101
8Identification of Imprinted Loci by Methylation: Use of Methylation-Sensitive Representational Difference Analysis (Me-RDA)113
9Ribonuclease Protection133
10Quantitative RT-PCR-Based Analysis of Allele-Specific Gene Expression145
11Allele-Specific In Situ Hybridization (ASISH)153
12RNA-FISH to Analyze Allele-Specific Expression169
13Flow Cytometry and FISH to Investigate Allele-Specific Replication Timing and Homologous Association of Imprinted Chromosomes181
14Southern Analysis Using Methyl-Sensitive Restriction Enzymes193
15A PCR-Based Method for Studying DNA Methylation205
16Bisulfite-Based Methylation Analysis of Imprinted Genes217
17Direct Analysis of Chromosome Methylation229
18In Vitro Methylation of Predetermined Regions in Recombinant DNA Constructs243
19In Vitro Methylation of Specific Regions in Recombinant DNA Constructs by Excision and Religation251
20Detection of Methyl-Sensitive DNA-Binding Proteins with Possible Involvement in the Imprinting Phenomenon259
21Probing Chromatin Structure with Nuclease Sensitivity Assays269
22Examining Histone Acetylation at Specific Genomic Regions285
23Purification of the McCP2/Histone Deacetylase Complex from Xenopus Iaevis297
24Reconstitution of Chromatin In Vitro309
25Genomic Imprinting in Plants327
Index371

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