Ward A. (Ed.). Genomic Imprinting: Methods and Protocols

Humana Press, 2002. - 389 p. - 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.Generation of Monoparental Embryos for Investigation into Genomic Imprinting
Deriving and Propagating Mouse Embryonic Stem Cell Lines for Studying Genomic Imprinting
Balanced Translocations for the Analysis of Imprinted Regions of the Mouse Genome
Production of YAC Transgenic Mice by Pronuclear Injection
A Transgenic Approach to Studying Imprinted Genes: Modified BACs and PACs
Methylation-Sensitive Genome Scanning
Subtraction-Hybridization Method for the Identification of Imprinted Genes
Identification of Imprinted Loci by Methylation: Use of Methylation- Sensitive Representational Difference Analysis (Me-RDA)
Ribonuclease Protection
Quantitative RT-PCR-Based Analysis of Allele-Specific Gene Expression
Allele-Specific In Situ Hybridization (ASISH)
RNA-FISH to Analyze Allele-Specific Expression
Flow Cytometry and FISH to Investigate Allele-Specific Replication Timing and Homologous Association of Imprinted Chromosomes
Southern Analysis Using Methyl-Sensitive Restriction Enzymes
A PCR-Based Method for Studying DNA Methylation
Bisulfite-Based Methylation Analysis of Imprinted Genes
Direct Analysis of Chromosome Methylation
In Vitro Methylation of Predetermined Regions in Recombinant DNA Constructs
In Vitro Methylation of Specific Regions in Recombinant DNA Constructs by Excision and Religation
Detection of Methyl-Sensitive DNA-Binding Proteins with Possible Involvement in the Imprinting Phenomenon
Probing Chromatin Structure with Nuclease Sensitivity Assays
Examining Histone Acetylation at Specific Genomic Regions
Purification of the MeCP2/Histone Deacetylase Complex from Xenopus laevis
Reconstitution of Chromatin In Vitro
Genomic Imprinting in Plants