Abstract
Chromosome conformation capture (3C) is a biochemical assay to reveal higher order chromosomal organizations mediated by physical contact between discrete DNA segments in vivo. Chromosomal organizations are involved in transcriptional regulation of a number of genes in various cell types. We have adapted 3C for analyzing the intrachromosomal looping organization of rod and cone photoreceptor genes in the mammalian retina. Here, we describe a detailed protocol for 3C assays on whole mouse retinas. Using the M-cone opsin gene as an example, we demonstrate how to genetically distinguish 3C signals from cones versus rods in retinal 3C assays. We also describe the challenges and key points of 3C design and performance as well as appropriate controls and result interpretations.
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Acknowledgment
We thank Dr. Anand Swaroop and Dr. Connie Cepko for providing Nrl −/− and Crx −/− mice, Hui Wang for technical assistance, and Anne Hennig for critical reading of the manuscript. This work was supported by NIH EY012543 (to SC), NIH EY02687 (to WU-DOVS), Lew Wasserman Merit Award (to SC), and unrestricted fund from Research to Prevent Blindness (to WU-DOVS).
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Peng, GH., Chen, S. (2012). Revealing Looping Organization of Mammalian Photoreceptor Genes Using Chromosome Conformation Capture (3C) Assays. In: Wang, SZ. (eds) Retinal Development. Methods in Molecular Biology, vol 884. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-848-1_22
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DOI: https://doi.org/10.1007/978-1-61779-848-1_22
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-848-1
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