Genomic Imprinting: Methods and Protocols / Edition 1

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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.

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Editorial Reviews

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"...a multi-authored volume in which the methods of gene imprinting are discussed in detail in 25 chapters. The prools are very detailed with multiple schemes and figures." - Journal of Pediatric Endocrinology and Metabolism
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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
  • Edition number: 1
  • Pages: 377
  • Product dimensions: 5.98 (w) x 9.02 (h) x 0.88 (d)

Table of Contents

1 Generation of Monoparental Embryos for Investigation into Genomic Imprinting 1
2 Deriving and Propagating Mouse Embryonic Stem Cell Lines for Studying Genomic Imprinting 21
3 Balanced Translocations for the Analysis of Imprinted Regions of the Mouse Genome 41
4 Production of YAC Transgenic Mice by Pronuclear Injection 55
5 A Transgenic Approach to Studying Imprinted Genes: Modified BACs and PACs 67
6 Methylation-Sensitive Genome Scanning 83
7 Subtraction-Hybridization Method for the Identification of Imprinted Genes 101
8 Identification of Imprinted Loci by Methylation: Use of Methylation-Sensitive Representational Difference Analysis (Me-RDA) 113
9 Ribonuclease Protection 133
10 Quantitative RT-PCR-Based Analysis of Allele-Specific Gene Expression 145
11 Allele-Specific In Situ Hybridization (ASISH) 153
12 RNA-FISH to Analyze Allele-Specific Expression 169
13 Flow Cytometry and FISH to Investigate Allele-Specific Replication Timing and Homologous Association of Imprinted Chromosomes 181
14 Southern Analysis Using Methyl-Sensitive Restriction Enzymes 193
15 A PCR-Based Method for Studying DNA Methylation 205
16 Bisulfite-Based Methylation Analysis of Imprinted Genes 217
17 Direct Analysis of Chromosome Methylation 229
18 In Vitro Methylation of Predetermined Regions in Recombinant DNA Constructs 243
19 In Vitro Methylation of Specific Regions in Recombinant DNA Constructs by Excision and Religation 251
20 Detection of Methyl-Sensitive DNA-Binding Proteins with Possible Involvement in the Imprinting Phenomenon 259
21 Probing Chromatin Structure with Nuclease Sensitivity Assays 269
22 Examining Histone Acetylation at Specific Genomic Regions 285
23 Purification of the MeCP2/Histone Deacetylase Complex from Xenopus laevis 297
24 Reconstitution of Chromatin in Vitro 309
25 Genomic Imprinting in Plants 327
Index 371
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