Femto-seq: Targeted photo-biotinylation, pulldown and sequencing of locus and region-specific DNA from femtoliter volumes within individual cells
Femto-seq:从单个细胞内的飞升体积中对位点和区域特异性 DNA 进行靶向光生物素化、下拉和测序
基本信息
- 批准号:10587721
- 负责人:
- 金额:$ 42.13万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-02-01 至 2027-01-31
- 项目状态:未结题
- 来源:
- 关键词:3-DimensionalArchitectureBiologicalBiological AssayBiotinBiotinylationCell LineCell NucleusCellsCellular StressChromatinCollectionComputer softwareComputers and Advanced InstrumentationConsumptionCustomCytosolDNADNA SequenceDNA sequencingDataDedicationsDetectionDiseaseDisease susceptibilityEnhancersEnsureEnvironmentEnvironmental Risk FactorEukaryotaExperimental DesignsFluorescent in Situ HybridizationGene ExpressionGene Expression RegulationGene SilencingGenesGenetic DiseasesGenomeGenomic SegmentGenomicsGoalsHandImageImage AnalysisIn SituIndividualKnowledgeLabelLasersLibrariesMachine LearningManualsMeasuresMethodsMicrofluidic MicrochipsMicrofluidicsMicroscopeMicroscopicMicroscopyMolecular ConformationNuclearNuclear StructureOpticsPhysiologic pulsePopulationPopulation AnalysisPreparationProceduresProcessProtocols documentationRegulationReportingResearch PersonnelResolutionRoleSamplingScanningSeriesShapesSignal TransductionSiteStem Cell ResearchStructureStructure/Function NucleiSystemTechnologyTimeTransgenesTreatment ProtocolsTubeValidationcellular imagingchromosome conformation capturecrosslinkdesigndesign and constructionexperimental studyflexibilitygene functiongenome-widegenomic locusgenomic toolsimage processingimaging modalityimprovedinsightinstrumentinterestmulti-photonnew technologynovelpromoterreal-time imagesresponsesample fixationsoftware developmenttechnology research and developmentthree dimensional structuretwo-photon
项目摘要
Changes in the spatial organization of the genome are directly involved in gene regulation during differentiation,
cellular stress responses and disease initiation. Existing approaches such as chromosome conformation capture
(3C) methods and DNA fluorescent in situ hybridization (DNA-FISH) have provided information on the overall 3D
structure of the nucleus at the chromatin level, providing critical insights into how our genomes are regulated.
Nonetheless, new technologies are needed to uncover the finer details on the role of nuclear architecture,
topological domains, and genomic interactions. Studies of gene regulation would benefit from a technology that
fills the niche between the ensemble averaged 3C methods and the single cell, low throughput DNA-FISH
approach. We have recently developed a novel optical technology we call “Femto-seq” that does just this – it
allows users to obtain DNA sequence information from targeted femtoliter volumes within the nucleus of selected
cells. Femto-seq provides a new way to examine genomic contacts near a specific gene locus or any nuclear
region of interest (e.g. nuclear bodies). Using 3D localized two-photon excitation, we can photochemically
biotinylate any region of the nucleus we can fluorescently label and identify in volumes which can be as small as
a ½ of a femtoliter. The process is carried out on a population of cells using a combined two-photon/confocal
microscope which images, locates fluorescently labeled regions of interest and then irradiates those regions
using 700 nm femtosecond pulses to biotinylate the chromatin by photochemically cross-linking the DNA with a
psoralen-biotin compound. Nuclei are isolated and the biotinylated DNA from the targeted region pulled down
and sequenced. Because the cells are imaged to locate the regions of interest, they can also be screened for
other parameters, allowing for the collection of targeted biotinylated DNA only from user selected cells within the
cell population, providing single-cell like genomic information from a sub-set of cells within the population. We
have proof-of-concept data from a cell line with a fluorescently labeled transgene we used as our targeted region,
and show that we can obtain DNA highly enriched in transgene locus sequences. The goals of this Focused
Technology Research and Development R01 project are to (Aim 1) design and construct a dual confocal/two-
photon microscope capable of targeting and irradiating user selected regions-of-interest in a population of cells
in a high-throughput automated fashion, (Aim 2) create a chromatin isolation pipeline based on novel microfluidic
designs to efficiently purify and prepare the DNA for sequencing, and (Aim 3) demonstrate the improvement
obtained from aims 1 and 2 in a series of Femto-seq experiments designed to produce quantitative metrics of
improvement and to uncover new knowledge on how environmental signals may be relayed through the cytosol
and into the nucleus. Femto-seq is a unique new way of investigating the spatial and regulatory relationships
between DNA sequences and any microscopically visible region-of-interest in the nucleus.
基因组空间组织的变化直接参与分化过程中的基因调控,
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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WARREN R ZIPFEL其他文献
WARREN R ZIPFEL的其他文献
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{{ truncateString('WARREN R ZIPFEL', 18)}}的其他基金
Broad wavelength range Zeiss 780 NLO/confocal system for the Cornell Imaging Core
适用于康奈尔成像核心的宽波长范围 Zeiss 780 NLO/共焦系统
- 批准号:
8734809 - 财政年份:2014
- 资助金额:
$ 42.13万 - 项目类别:
A miniature confocal for long term 3D multicolor imaging within a C02 incubator
用于 CO2 培养箱内长期 3D 多色成像的微型共焦
- 批准号:
8575684 - 财政年份:2013
- 资助金额:
$ 42.13万 - 项目类别:
Zeiss 510 Multiphoton/single-photon Confocal Microscope
Zeiss 510 多光子/单光子共焦显微镜
- 批准号:
6877387 - 财政年份:2005
- 资助金额:
$ 42.13万 - 项目类别:
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