遗传学实验技术

Chromatin immunoprecipitation (ChIP) is an invaluable method to study the specific interaction of regulatory proteins with genomic DNA. Since its first development, it has been modified extensively to make it applicable to many different cell types and experimental systems. The cro ...

Histone lysine and arginine methylation involved in gene activation and silencing is dynamically regulated. However, partly limited to the research technologies previously available, the dynamics of global histone methylation on a site-specific basis have not been fully pursu ...

Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged W ...

In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associ ...

Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be inve ...

Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, w ...

This review concisely highlights the complexity of regulatory events. It provides examples of how interconnectivity of regulatory hubs could maintain transcriptional synergy and orchestrate the proper spatial and temporal patterns of gene expression.

Identification and verification of novel transcription factor interactions is an inherent step in the discovery of molecular mechanisms driving gene transcription and regulation. Co-immunoprecipitation and GST-pull down are often key techniques in the verification proc ...

Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can app ...

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiati ...

Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its reco ...

Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c�hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of c ...

Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics me ...

In traditional electrophoresis mobility shift assay (EMSA) a single 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. ...

Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interact ...

A comprehensive identification of protein–DNA interactions that drive processes such as transcription and replication, both in prokaryotic and eukaryotic organisms, remains a major technical challenge. In this chapter, we present a SILAC-based DNA affinity purification met ...

The yeast one-hybrid system is a powerful genetic method to identify DNA–protein interactions, but there is a major limitation inherent to the system. Namely, frequency of false positives generated by yeast endogenous transcription factors has been thought to be higher than that of true po ...

More than 20 years have passed since the advent of genetic manipulation of the mouse germline using cultures of pluripotent embryonic stem cells. Still, despite remarkable successes in the mouse, the application of stem cell cultures for transgenesis in other mammalian species has been co ...

Transposable elements represent a class of DNA molecules that have the ability to move genetic material from one location to another. Since the first recognition and description of their activities by Barbara McClintock beginning in the 1950s, researchers have been harnessing their mo ...

In this chapter, we will discuss the use of lentiviral vectors to generate transgenic animals. Specifically, we will review the technology as applied to the generation of rodents and birds. But many of the procedures can be applied in other species. We review the production of vectors, their design a ...