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Global Tracing of Epigenetic Marks in Early C. elegans Embryos

早期线虫胚胎表观遗传标记的全局追踪

基本信息

批准号：
7991075
负责人：
Robert Riehn
金额：
$ 21.13万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7991075
关键词：
Antibodies Caenorhabditis elegans Caliber Cell Line Cell division Cells Chromatin Chromosomes Comb animal structure Complex Coupled Cytolysis DNA Detection Development Devices Embryo Embryonic Development Epigenetic Process Evolution Fiber FISH Figs - dietary Fluorescence Fluorescence Microscopy Fluorescent Antibody Technique Generations Genes Genome Genomics Hand Histones Image In Situ Hybridization Individual Individuality Label Malignant Neoplasms Maps Measurement Mechanics Methods Microfluidics Mitotic Mitotic Chromosome Modification Molecular Monitor Organ Organism Organogenesis Pathway Analysis Pattern Preparation Process Proteome Public Health Regulatory Element Resolution Signal Transduction Staging Stretching Structure Structure of thyroid parafollicular cell Surveys System Tail Techniques Testing Tissues Whole Organism Work base cancer stem cell design embryo cell epigenomics genome-wide histone modification improved micro-total analysis system nanochannel nanofluidic novel programs public health relevance tool

项目摘要

DESCRIPTION (provided by applicant): Epigenetic factors participate in the development and organogenesis in higher organisms. For the underlying process of differentiation, the emergence of epigenetic individuality of cells within the organism is required. Our application aims at developing a method for preparing global profiles of histone modification states over whole chromosomes, from single cells. The measurement of epigenetic individuality also becomes important in understanding organs undergoing rapid change, such as cancers that include cancer stem cells. The proposed method is conceptually similar to fiber-FISH. In particular, we plan on extracting chromosomes from a single, targeted cell. We then plan on visualizing the histone modification pattern by stretching whole chromosomes and fluorescent imaging of antibodies coupled to those histone tail modifications. In order to provide homogeneous stretching of ultra-long chromatin molecules, we will utilize a novel stretching technique based on nanofluidic channels. We have recently shown an elongation of 49 kbase chromatin molecules in such a channel to about 3 micrometers, leading to an equivalent expected resolution 6 kbase under fluorescence microscopy. The use of nanochannels enables stretching of ultra-long molecules without the problem of tension- based breaking such as in molecular combing. We propose to demonstrate the utility of our approach by following the global epigenetic state of early C. elegans embryos. Since C. elegans is an organism with an extremely early commitment of some cells to specific cell lines, we anticipate individuality of cells within such embryos. On the other hand, we will also be able to probe whether pluripotent cells are identical, or are also characterized by some individuality. All preliminary work will also be performed on the embryonic C. elegans system. Our application is structured into three distinct parts. Part one investigates whether whole chromosomes derived from mitotic cells can be stretched inside nanochannels. Part two is concerned with the design, development, and testing of a microfluidic platform that enables automated lysis of embryos and cells, and introduction into nanochannels. Part three will follow the epigenetic embryonic development from the 8 to the 28 cell stage. PUBLIC HEALTH RELEVANCE: This project has the potential to improve public health by developing a new technique for monitoring the epigenetic programming state of genomic sized molecules, ideally whole chromosomes, from single cells. Epigenetic programming has broad implications critical to both diseased and healthy states, in particular cancer development. This proposed work will enable tracing the epigenetic individuality of single cells from complex tissues.

描述（由申请人提供）：