Genomic Imprinting

Genomic imprinting is the process by which gene activity is regulated according to parent of origin. Usually, this means that either the maternally inherited or the paternally inherited allele of a gene is expressed while the opposite allele is repressed. The phenomenon is largely restricted to mammals and flowering plants and was first recognized at the level of whole genomes. Nuclear transplantation experiments carried out in mice in the late 1970s established the non-equivalence of the maternal and paternal genomes in mammals, and a similar conclusion was drawn from studies of interploidy crosses of flowering plants that extend back to at least the 1930s. Further mouse genetic studies, involving animals carrying balanced translocations (reviewed in Chapter 3), indicated that imprinted genes were likely to be widely scattered and would form a minority within the mammalian genome. The first imprinted genes were identified in the early 1990s; over forty are now known in mammals and the list continues steadily to expand.

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Klappentext
Imprinted genes, many of which generally control growth and development, frequently lose their imprints during cancer progression, a loss that then plays a substantial role in uncontrolled tumor growth. Imprint instability also appears to be a major limitation to the success of mammalian cloning experiments. In Genomic Imprinting: Methods and Protocols, 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. The majority of protocols describe molecular techniques that allow examination of gene structure or expression in an allele-specific manner. 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.
Incorporating a wealth of knowledge from leading exponents in the field, Genomic Imprinting: Methods and Protocols brings together all the essential molecular, genetic, and embryological methods commonly used in today's laboratories for the identification and analysis of imprinted genes.

Inhalt
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.- Methylation-Sensitive Genome Scanning.- Subtraction-Hybridization Method for the Identification of Imprinted Genes.- Identification of Imprinted Loci by Methylation.- 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 Acetlylation at Specific Genomic Regions.- Purification of the MeCP2/Histone Deacetylase Complex from Xenopus laevis.- Reconstitution of Chromatin In Vitro.- Genomic Imprinting in Plants.