GENETICALLY ENCODED SPARSE LABELING AND EXPRESSION FOR IN VIVO STUDIES

用于体内研究的基因编码稀疏标记和表达

基本信息

批准号：
8904738
负责人：
MICHAEL L NONET
金额：
$ 7.63万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8904738
关键词：
Animals Applications Grants Automobile Driving Biological Biological Models Cell Transplantation Cell physiology Cells Cellular Structures Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Code Complex DNA DNA Repair Development Disease Dyes Elements Estrogen Receptors Event Figs - dietary Frequencies Generations Genetic Genetic Recombination Genetic Screening Golgi Apparatus Guide RNA Health Human Image Immigration Individual Injection of therapeutic agent Label Life Ligand Binding Domain Ligands Mediating Methods Mitotic Modeling Molecular Genetics Mus Muscle Natural regeneration Neurons Nonhomologous DNA End Joining Nuclear Organism Pharmaceutical Preparations Plasmids Play Population Positioning Attribute Preclinical Drug Evaluation Process Proteins RNA Polymerase II RNA Polymerase III Reporter Reporter Genes Role Sampling Shapes Site Somatic Cell Staining method Stains Structure System Tamoxifen Techniques Terminator Codon Testing Time Tissues Transgenes Transgenic Animals Transgenic Organisms Vertebrates Virus Diseases YAC Clone Zebrafish adult stem cell base cell behavior cell type density design ds-DNA gene function genetically modified cells human disease human tissue in vivo interest man neuron development nuclease promoter repaired site-specific integration targeted sequencing theories therapy design tissue fixing tool

项目摘要

DESCRIPTION (provided by applicant): Sparse labeling of experimental tissue has been a powerful approach for cell biological analysis from the time of Ramon y Cajal, who used Golgi staining to describe in great detail the structure of neuronal tissue. To this date such labeling i critical for the analysis of neurons in whole tissue as the density and complexity of axonal and dendritic processes make tracing individual cells virtually impossible in ubiquitously labeled samples. Sparse labeling techniques also play critical roles in examining the behavior of cells during migration, in determining lineage relationships of cells, and assessing the cell autonomy of gene function. A variety of approaches have been used to sparsely label samples both in fixed and living tissue including Golgi staining, single cell dye injections, transient expression f DNA constructs, viral infection and cell transplantation. In vertebrate systems, several transgenic approaches to sparsely label cells have also been developed including use of promoters that display extensive variegation in expression, drug inducible activation of promoters and stimulation of mitotic interchromosomal recombination. However, none of these approaches for obtaining sparse labeling are easily applied for large-scale analysis in vertebrates (genetic screens or drugs screens). We propose to develop a robust, reliable method to create transgenic animals that will sparsely label a cell population and be readily reproduced from generation to generation. Conceptually, the idea is to use a newly described CRISPR/Cas9 site-specific nuclease to drive non-homologous end joining DNA repair in select cells to modify a non-expressed GFP reporter into one that expresses. If successful the approach should be widely applicable for the analysis of neuronal structure (and other cell types also) in mouse or zebrafish models of human neuronal diseases.

描述（由申请人提供）：从 Ramon y Cajal 时代起，实验组织的稀疏标记一直是细胞生物学分析的有力方法，他使用高尔基染色来详细描述神经元组织的结构。迄今为止，这种标记对于分析整个组织中的神经元至关重要，因为轴突和树突过程的密度和复杂性使得在普遍标记的样本中追踪单个细胞几乎是不可能的。稀疏标记技术在检查细胞迁移过程中的行为、确定细胞的谱系关系以及评估细胞基因功能的自主性方面也发挥着关键作用。多种方法已被用于对固定组织和活体组织中的样品进行稀疏标记，包括高尔基体染色、单细胞染料注射、瞬时表达 DNA 构建体、病毒感染和细胞移植。在脊椎动物系统中，还开发了几种稀疏标记细胞的转基因方法，包括使用表达广泛多样化的启动子、启动子的药物诱导激活和刺激有丝分裂染色体间重组。然而，这些获得稀疏标记的方法都不能轻易应用于脊椎动物的大规模分析（遗传筛选或药物筛选）。我们建议开发一种稳健、可靠的方法来创建转基因动物，这些动物将稀疏地标记细胞群并易于代代繁殖。从概念上讲，这个想法是使用一种新描述的 CRISPR/Cas9 位点特异性核酸酶来驱动选定细胞中的非同源末端连接 DNA 修复，将不表达的 GFP 报告基因修饰为表达的报告基因。如果成功，该方法应广泛适用于人类神经元疾病的小鼠或斑马鱼模型中神经元结构（以及其他细胞类型）的分析。