The Role of Alternative Splicing Factor Sfrs10 in Neural Development

选择性剪接因子 Sfrs10 在神经发育中的作用

基本信息

批准号：
8420479
负责人：
RAHUL N KANADIA
金额：
$ 17.29万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8420479
关键词：
Address Affect Alternative Splicing Amacrine Cells Antibodies Architecture Bioinformatics Biological Assay Biological Models Birth Order Brain Cell Separation Cells Central Nervous System Part Code Complementary DNA Complex Connecticut Data Defect Development Disease Electroporation Embryo Event Exclusion Exons Fluorescence Gene Expression Profiling Genes Glass Goals Grant House mice Human Immunofluorescence Immunologic In Situ Hybridization Infection Instruction Knockout Mice Laboratories Lead Left Link Messenger RNA Methods Microarray Analysis Motor Neurons Mus Mutation Myotonic Dystrophy Needles Neuraxis Neuroglia Neurons Nonsense Codon Northern Blotting Pathogenesis Pathway interactions Pattern Phenotype Plasmids Play Positioning Attribute Process Production Protein Isoforms Proteins Proteome RNA RNA Interference RNA Splicing Reporting Research Resolution Retina Retinal Retinitis Pigmentosa Reverse Transcriptase Polymerase Chain Reaction Role Science Spinal Muscular Atrophy Stem cells Techniques Tissues Tretinoin Universities Vertebrates Work base career cell type deep sequencing design gain of function ganglion cell glutamate receptor type B human fetus tissue imprint in vivo indexing injured insight nervous system development neurodevelopment postnatal professor relating to nervous system research study retinal damage retinal progenitor cell

项目摘要

Alternative .splicing allows hutTians to protiuce a vast proteome diversity from a relatively few (~24,000) protein coding genes. This ROO proposal is part: of an overall objective to gain insight into the fole of alternative splicing in retinal development. 1 have chosen to address this question by focusing on SfrslO, with the specific hypothesis that it is essential for cell fate determination and differentiation. The overall goal is to determine the expression, function and targets of SfrslO during retinal development. I have employed in situ hybridization, immunofluorescence and microarray analysis to determine the expression of SfrslO during retinal development. In all, SfrslO appears to be expressed in progenitor cells and differentiating amacrine cells. Interestingly, the microarray data obtained from single retinal cells shows that certain progenitor cell markers such as cycling Dl and Sfrsp2 are enriched along with the expression of SfrslO. To determine the function of SfrslO,! have employed in vivo electroporation to deliver F^NAi or the misexpression construct in PO retinal progenitor cells. 1 have found that loss of SfrslO function results in increase in the numberof Mullerglia at the expense of neurons. This experiment utilizes a Hamilton needle to deliver the plasmid into the subretinal space. However, this method has often created damage to the retina. 1 have now employed a different method which utilized glass needles that leave a negligible imprint on the retina. While RNAi constructs reported in the grant have been used to determine the function of SfrslO, a conditional knockout mouse is crucial to this proposal. The production of such a mouse has been initiated at the University of Connecticut, where I will begin my position as an assistant professor on August I'S 2010. As 1 continue this project in my laboratory at the University of Connecticut, I will further refine the expression of SfrslO, perform gain and loss of SfrslO function with the electroporation techinque. Finally, the conditional knockout mouse will be crossed to retina specific Cre lines followed by detailed analysis of the phenotype. Also, the knockout mouse retinal cells will be used for deep (454) sequecing to identify the targets of SfrslO. Extenstion of this work in my laboratory will provide me with preliminary data that 1 intend to use for an ROl grant that should launch my independent career in science.

替代性。拼图允许huttians从相对较少的（约24,000）蛋白质中吸收巨大的蛋白质组多样性编码基因。此ROO提案是一部分：一个总体目标，以深入了解替代剪接的功能在视网膜发展中。 1选择通过专注于SFRSLO来解决这个问题假设它对于细胞命运的确定和分化至关重要。总体目标是确定视网膜发育过程中SFRSLO的表达，功能和靶标。我曾经使用原位杂交，免疫荧光和微阵列分析以确定视网膜发育过程中SFRSLO的表达。总的来说，SFRSLO似乎在祖细胞中表达并分化了无长束细胞。有趣的是，从单个视网膜细胞获得的微阵列数据表明，某些祖细胞标记（例如循环DL） SFRSPL与SFRSLO的表达一起富含SFRSP2。要确定SFRSLO的功能！有在体内电穿孔中使用，以在Po视网膜祖细胞中传递F^nai或Misexpression构建体。 1 已经发现SFRSLO功能的丧失导致Mullerglia的数量增加，而神经元的代价。该实验利用汉密尔顿针将质粒输送到视网膜下空间中。但是，这个方法经常对视网膜造成损害。 1现在使用了一种使用玻璃的不同方法在视网膜上留下微不足道的烙印的针头。虽然赠款中报道的RNAi结构是用于确定SFRSLO的功能的条件基因敲除小鼠用于该建议至关重要。这这种鼠标的生产已在康涅狄格大学开始，我将开始自己的立场一位助理教授于2010年8月1日。当1继续在我的大学实验室继续这个项目。康涅狄格州，我将进一步完善SFRSLO的表达，执行SFRSLO功能的增益和丧失电穿孔Techinque。最后，有条件的敲除鼠标将越过视网膜特定的CRE线然后对表型进行详细分析。此外，敲除小鼠视网膜细胞将用于深（454）序列以识别SFRSLO的目标。这项工作在我的实验室中的延伸将为我提供 1打算用于ROL赠款的初步数据，该数据应启动我的科学独立职业。