Jun 7, 2014

Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins

BioRxiv : the Preprint Server for Biology
Toshitsugu Fujita, Hodaka Fujii

Abstract

Background Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo . We have developed the insertional chromatin immunoprecipitatin (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward. Results In this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal, the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. 3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could effic...Continue Reading

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Mentioned in this Paper

Study
In Vivo
Genome
Baculovirus Expression System
Isolation Aspects
Paired box 5 protein (B-cell lineage specific activator)
Molecular Probe Techniques
Proteins, Recombinant DNA
Bombyx
Reverse Transcriptase Polymerase Chain Reaction

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