ArticleBMC molecular biology2014
Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.
Article in BMC molecular biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed, 31 citations in OpenAlex.
- Review
- Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations.Communications biology · 2021Article
- Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexesBiology methods & protocols · 2021Article
- CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.Scientific reports · 2019Article
- Article
- Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line.DNA research : an international journal for rapid publication of reports on genes and genomes · 2017Article
- Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP.Journal of visualized experiments : JoVE · 2016Article
- Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies.Gene regulation and systems biology · 2016Review
- Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research.International journal of molecular sciences · 2015Review
- Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins.International journal of molecular sciences · 2015Review
- Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.PloS one · 2015Article
- A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.PloS one · 2015Article
- Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.PloS one · 2014Article
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2 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundComprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (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 than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed.
resultsIn this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal (NLS), the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR.
conclusionsThe iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions as compared to the previously published iChIP protocol.
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