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：
8823435
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/8823435
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 gene function genetically modified cells human disease human tissue in vivo interest man neuron development nuclease promoter public health relevance repaired site-specific integration 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）：从拉蒙·凯尔（Ramon Y Cajal）开始，实验组织的稀疏标记一直是细胞生物学分析的强大方法，他们使用高尔基染色来详细描述神经元组织的结构。到目前为止，这种标记对于整个组织中神经元的分析至关重要，因为轴突和树突过程的密度和复杂性使得在无处不在标记的样品中几乎不可能追踪单个细胞。稀疏标记技术在检查迁移过程中细胞的行为，确定细胞的谱系关系以及评估基因功能的细胞自主权方面也起着关键作用。已使用多种方法将样品稀疏地标记在固定组织和活性组织中，包括高尔基染色，单细胞染料注射，瞬时表达F 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，包括使用启动子，这些启动子在表达中显示出广泛的变化，启动子的药物诱导激活以及刺激有丝分裂的染色体染色体重组。然而，这些用于获得稀疏标记的方法均不容易在脊椎动物（遗传筛查或药物筛查）中进行大规模分析。我们建议开发一种可靠，可靠的方法来创建转基因动物，该动物将稀疏地标记细胞群，并很容易代代相传。从概念上讲，这个想法是使用新描述的CRISPR/CAS9特异性核酸酶来驱动与选择细胞中DNA修复的非同源末端，以将非表达的GFP报告基因修改为表达的一个。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。