Sludge(R1-AGS/R2-AGB/R3-eAGB/R3-iAGB)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1155813 | ENA |
REPOSITORIES: ENA
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Project description:We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2 and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We inserted STITCH into five different positions of the remaining allele of the locus: \\"STITCH+30kb\\", \\"STITCH+440kb\\", \\"STITCH+1760kb\\" and \\"STITCH+1790kb\\" have the STITCH insertions away from the MYC promoter for the indicated distances to the telomeric side of the p arm of the chromosome. \\"STITCH-30kb\\", at the 30-kb upstream from the MYC. We also made a deletion clone of the enhancer region, termed del(30-440). We made deletion of each CTCF array, L (delL) and R (delR), inversion of R (invR), deletion of the middle five binding sites from L2 to R2 (del(L2-R2)), and deletion of the six sites but for R3 (del(L1-R2)) in STITCH+30kb. We also obtained deletion and inversion of the whole of STITCH (del(L1-R3) and inv(L1-R3)). We integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH+30kb clone (STITCH/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the MYC promoter as a viewpoint to see how STITCH impacts on the chromatin conformation.
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