A toolkit to reversibly disrupt nuclear bodies and move genes among compartments
可逆地破坏核体并在区室之间移动基因的工具包
基本信息
- 批准号:9134116
- 负责人:
- 金额:$ 44万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2015
- 资助国家:美国
- 起止时间:2015-09-01 至 2018-08-31
- 项目状态:已结题
- 来源:
- 关键词:AffectAlgorithmsAllelesAuxinsBindingBinding ProteinsBiologicalCell CycleCell NucleolusCell divisionCellsChimeric ProteinsChromatinChromosome TerritoryChromosomesClustered Regularly Interspaced Short Palindromic RepeatsCodeCollaborationsCommunitiesDevelopmentDiseaseEngineeringEpigenetic ProcessExhibitsGene ExpressionGene TargetingGene-ModifiedGenesGenomicsGoalsHealthHeterochromatinHourIndividualKineticsKnock-outLabelLocationMeasuresMethodsMolecularMonitorMovementMusNuclearNuclear RNAPattern RecognitionPeripheralPlayProteinsQuality ControlRNA InterferenceReportingResearch PersonnelRiskRoleRunningSeedsSiteStructural GenesSystemTechniquesTechnologyTestingTetracyclinesTranscription CoactivatorUndifferentiatedValidationbaseblastomere structurecell typeembryonic stem cellhomologous recombinationinterestknock-downmembernovelnucleaseresearch studytooltool development
项目摘要
DESCRIPTION (provided by applicant): The proposal goal is to create a toolkit in which endogenous nuclear bodies (NBs) and chromatin compartments can be reversibly disrupted and endogenous genes can be moved among compartments. This toolkit will allow study of the functional interactions between NBs and chromatin and will be made widely available to the scientific community through the 4D Nucleome consortium. It is clear that chromatin reorganizes during differentiation, yet it is not well-understood how NBs interact with chromatin and influence
this reorganization. For example, it has been difficult to study how organization of the repressive
chromatin compartment might be seeded or affected by NBs such as the nucleolus because current tools are primarily limited to the study of exogenously expressed components and/or are not rapidly reversible. This toolkit will be developed in mouse embryonic stem cells (mESCs) because they are primary cells that can be differentiated into multiple cell types by end users. In
Specific Aim 1, auxin inducible degrons (AIDs) will be employed to construct systems in which NBs and chromatin compartments are rapidly and reversibly disrupted. The AID sequence will be stably integrated into both alleles of endogenous genes coding for A) proteins essential for the integrity of the nucleolus (as a test NB) and B) lamina proteins that anchor peripheral heterochromatin (PH, as a test compartment). In contrast to current RNAi knockdown or cre/lox-based knockout techniques, this approach will allow rapid degradation of target proteins (minutes/hrs vs days), is not dependent on the cell cycle and is rapidly reversible. In Specific Aim 2, a novel modified TetO/TetR system will be used to tether specific proteins of interest to individual genes and move genes among compartments. Tethering of specific nucleolar and laminar structural proteins to both active and inactive mESC genes will be used as test cases and proteins that most effectively move genes among compartments will be identified. This tethering system will be more useful than current systems because it will not affect the chromatin context of the target region (as do large repetitive arrays) nor risk off-target effects (as in nuclease deficient dCas9-based approaches). Labeling unique genes is also simpler and quicker as compared to the transcription activator-like effector (TALE) approach, where TALEs targeting multiple sequences must be synthesized. In Specific Aim 3 we describe proof of principle experiments in differentiating cells to test the redundancy of the heterochromatic compartment and characterize microdomains within the PH. We demonstrate by collaboration that other researchers are interested in these tools. Successful implementation of these tools by the general scientific community would permit detailed study of the role that NBs play in chromatin organization and provide tethering tools to study how genes move between nuclear compartments during both development and disease.
描述(由申请人提供):该提案的目标是创建一个工具包,其中内源核体(NB)和染色质区室可以被可逆地破坏,内源基因可以在区室之间移动。该工具包将允许研究 NB 之间的功能相互作用。和染色质,并将通过 4D Nucleome 联盟向科学界广泛提供。 很明显,染色质在分化过程中会重组,但事实并非如此。充分了解 NB 如何与染色质相互作用并产生影响
例如,很难研究这次重组是如何组织的。
染色质区室可能会受到 NB(如核仁)的影响,因为当前的工具主要限于外源表达成分的研究和/或不能快速可逆。该工具包将在小鼠胚胎干细胞 (mESC) 中开发,因为它们是可以被最终用户分化成多种细胞类型的原代细胞。
具体目标 1,将采用生长素诱导型降解决定子 (AID) 构建系统,其中 NB 和染色质区室被快速且可逆地破坏。AID 序列将稳定整合到编码 A) 蛋白质完整性所必需的内源基因的两个等位基因中。核仁(作为测试 NB)和 B)锚定外周异染色质的核纤层蛋白(PH,作为测试隔室)。敲除或基于 cre/lox 的敲除技术,这种方法将允许目标蛋白快速降解(分钟/小时 vs 天),不依赖于细胞周期,并且快速可逆。在 Specific Aim 2 中,这是一种新型修饰的 TetO/TetR。系统将用于将感兴趣的特定蛋白质束缚在单个基因上,并在区室之间移动基因。将特定核仁和层状结构蛋白质束缚在活性和非活性 mESC 基因上将用作测试用例和蛋白质。将确定最有效地在区室之间移动基因的系统将比现有系统更有用,因为它不会影响目标区域的染色质环境(如大型重复阵列),也不会产生脱靶效应的风险(如在核酸酶中)。与转录激活因子样效应器 (TALE) 方法相比,标记独特基因也更简单、更快捷,在特定目标 3 中,必须合成针对多个序列的 TALE。我们描述了分化细胞的原理证明实验,以测试异染色质区室的冗余性并表征 PH 内的微域。我们通过合作证明,其他研究人员对这些工具感兴趣,一般科学界将允许详细说明。研究 NB 在染色质组织中发挥的作用,并提供束缚工具来研究基因在发育和疾病期间如何在核区室之间移动。
项目成果
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{{ truncateString('MARK T GROUDINE', 18)}}的其他基金
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