iChIP / enChIP FAQs
Questions
Q1. Where to insert LexA-binding elements?
Q2. How to insert LexA-binding elements?
Q3. How many LexA-binding elements should be inserted in a locus?
Q4. How much is the yield of purification of specific genomic regions?
Q5. Are LexA-binding elements stably retained in E. coli and cell lines?
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Q1. Where to insert LexA-binding elements?
A. Insertion of LexA-binding elements may affect chromatin structure such as nucleosome positioning and abrogate normal genome activities such as gene expression. Although the effects of insertion need to be tested empirically for each locus, the author's group has guidelines to avoid potential aberrant effects caused by insertion of LexA-binding elements. (a) For analysis of promoter regions near transcription start sites (TSSs), the insertion site should be several hundred base 5' to the transcription start site so that the insertion would not inhibit transcription or disrupt nucleosome positioning. In contrast, for identification of binding molecules of genomic regions with distinct boundaries such as enhancer or silencer, the LexA-binding elements can directly be juxtaposed to the regions because of less probability of inhibition of their function. (b) The insertion site should not be conserved among species because conserved regions are often binding sites of conserved binding molecules.
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Q2. How to insert LexA-binding elements?
A. For mouse embryonic stem cells, conventional gene targeting would be a choice. However, knocking-in into the genome of cultured cell lines has been more difficult. In this regard, advent of zinc-finger nucleases (ZFN), TALEN, and CRSPR/Cas9 technologies makes gene targeting much easier in cultured cell lines.
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Q3. How many LexA-binding elements should be inserted in a locus?
A. We use 8xLexA-binding elements for iChIP. By combining this with the second-generation tagged LexA DNA-binding domain, 3xFNLDD, we consistently purify 5 - 15% input genomic DNA. Since the yield is reasonably good, we don't think further increase in the number of LexA-binding elements is necessary. However, if you need to increase the number, we suggest you to keep the head-to-tail orientation of the LexA-binding elements. Our 8xLexA-binding elements are incomplete palindrome to be kept in E. coli. Complete palindrome would be lost in E. coli culture. Therefore, if you put the 8xLexA-binding elements in the reverse orientation, it would be difficult to get the correct clones.
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Q4. How much is the yield of purification of specific genomic regions?
A. We consistently purify 5 - 15% input genomic DNA by using the second-generation tagged LexA DNA-binding domain, 3xFNLDD and 8xLexA-binding elements.
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Q5. Are LexA-binding elements stably retained in E. coli and cell lines?
A. Our collaborators and we have observed higher mutation rates of LexA BE sequences in E. coli and target cells. Therefore, it would be safer to sequence them, at least, before and after transfection into cells. If possible, sequence confirmation should be done in each step of construction of LexA BE-containing plasmids including transformation into E. coli.