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. 2014 Oct;141(20):4018–4030. doi: 10.1242/dev.115709

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Experimental approach and validation. (A) Structure of the TALE-expressing piggyBac construct. TALE cDNA consists of the TALE sequence followed by a nuclear localisation domain (NLS), a VP64 domain, 2A (peptide sequence cleaved after translation) and mCherry fluorescent protein. TALE cDNA was cloned downstream of a tetracycline-responsive promoter (TetR), and within piggyBac long terminal repeats (LTRs) for stable transposase-mediated genomic integration. The DNA-binding domain (DBD) within the TALE sequence consists of twenty monomers. Monomers contain two hypervariable amino acids that determine nucleotide-binding specificity: NN, NI, NG or HD. (B,C) Schematics of the mouse Scl (Tal1) (B) and PU.1 (Spi1) (C) genomic loci, with the Scl+40kb and PU.1-14kb elements highlighted in green. TALE target sites within conserved (between human and mouse) sequences are highlighted in red. (D) Experimental approach to express TALEs in cell lines. K562 and 416B cells were co-transfected with the TALE-expressing piggyBac (TALE-PB) from A, a constitutively expressing rtTA piggyBac vector (pCAG-rtTA-PB) and a piggyBac transposase, to create inducible TALE-expressing cells. (E) Effect of expressing TALE-VP64 targeting Scl+40kb (T-VP64-Scl+40) in human K562 (left) and mouse 416B (right) cells on neighbouring gene expression. T-VP64-Scl+40 was expressed for 48 h by addition of doxycycline (dox) and gene expression in mCherry⁺ cells was determined relative to mCherry⁻ control cells. Error bars indicate s.d. of technical triplicates, and are representative of two biological replicates. (F) As in E, but for TALE-VP64 targeting PU.1-14kb (T-VP64-PU.1-14). (G) ChIP approach for TALE-VP64 proteins in H. An HA affinity tag was inserted at the N-terminus of the TALE-VP64 (HA-T-VP64); 416B cells were co-transfected as in D, sorted and ChIP performed 48 h after dox addition. (H) ChIP-qPCR enrichment of HA-tagged TALE-VP64 (HA-T-VP64) relative to IgG in HA-T-VP64-Scl+40 (pink), HA-T-VP64-PU.1-14 (red) and untransfected 416B control (green) cells at Scl+40kb, PU.1-14kb and a control region on chromosome 1 (Chr1). Error bars indicate s.d. of technical triplicates from one biological experiment.