遗传学实验技术

It is becoming increasingly apparent that chromatin is not randomly folded into the nucleus, but instead is highly organized into specific conformations within the nucleus. One consequence of such higher order structure is that chromatin looping can bring together genomic elements w ...

Imprinted genes are marked by parental allele specific DNA methylation and histone modifications which regulate their monoallelic expression. Chromatin immunoprecipitation (ChIP) is the technique of choice to characterize the histones associated with either maternal or p ...

Gene targeting in embryonic stem (ES) cells coupled with the site-specific Cre/loxP recombination system offers unique opportunities to identify and analyze the roles of cis-acting sequences in the regulation of imprinted gene expression. Although several different approach ...

This chapter serves as an introduction to the collection of genome-wide sequence and epigenomic data, as well as the use of these data in training generalized linear models (glm) to predicted imprinted status. This is meant to be an introduction to the method, so only the most straightforward exam ...

Enabled by high-throughput technologies that are capable of generating millions of sequencing reads, transcriptome sequencing is emerging as an important approach for mapping allelic imbalance (AI), where transcription is biased toward one allele in a diploid system. AI is identi ...

I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA m ...

Primordial germ cells (PGCs) play essential roles in both reproduction and development. In this chapter, we describe a method used in our laboratory for the immunopurification of PGCs from the mouse embryo. After dissection and disruption of the fetal gonad, PGCs are identified by a monoclon ...

The isolation and culture of both embryonic and extraembryonic stem cells provide an enormous opportunity to study the molecular processes that establish and maintain lineage-specific, monoallelic patterns of gene expression. This chapter describes the isolation an culture of ...

The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challe ...

Live-cell microscopy has demonstrated that many nuclear proteins bind transiently to target sites in chromatin. These binding interactions can be detected and quantified by two related live-cell imaging techniques, Fluorescence Correlation Spectroscopy (FCS) and Temporal ...

Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nuclear protein binding to chromatin in live cells. FRAP can be applied qualitatively to assess if chromatin binding interactions are altered by various biological perturbations. It can a ...

A vast body of evidence in the literature indicates that nucleosomes can act as barriers to transcriptional initiation. The nucleosome at the promoter inhibits association of transcription factors disallowing active transcription of the gene. We have found a nucleosome on tobacco pa ...

Histone proteins are the major protein components of chromatin – the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demons ...

To study the functions of histone modifications in the context of chromatin, it is necessary to be able to prepare nucleosomal templates that carry specific posttranslational modifications in a defined biochemical system. Here, we describe two sets of protocols for reconstituting de ...

ATP-dependent remodeling factors regulate chromatin structure by catalyzing processes such as nucleosome repositioning or conformational changes of nucleosomes. Predominantly, their enzymatic properties have been investigated using mononucleosomal substra ...

Genome-wide nucleosome mapping in vivo highlighted the extensive degree of well-defined nucleosome positioning. Such positioned nucleosomes, especially in promoter regions, control access to DNA and constitute an important level of genome regulation. However, the molecul ...

Regulatory mechanisms underlying eukaryotic gene expression, and many other DNA metabolic pathways, are tightly coupled to dynamic changes in chromatin architecture in the nucleus. Activation of gene expression generally requires the recruitment of histone acetyltransf ...

Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent n ...

ATP-dependent nucleosome-remodeling motors use the energy of ATP to alter the accessibility of the underlying DNA. Understanding how these motors alter nucleosome structure can be aided by following changes in histone–DNA contacts in real time. Here, we describe a fluorescence reson ...

A mutant library consisting of hundreds of designed point and deletion mutants in the genes encoding Saccharomyces cerevisiae histones H3 and H4 is described. Incorporation of this library into a suitably engineered yeast strain (e.g., bearing a reporter of interest), and the validation of ...