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

• 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
• 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

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.

替代剪接使HutTians能够从相对较少(约24,000)的蛋白质中产生巨大的蛋白质组多样性 编码基因。这项Roo提案是一个总体目标的一部分，该目标旨在深入了解替代剪接的好处 在视网膜发育过程中。我选择通过关注SfrslO来解决这个问题，具体 假设它对细胞命运的决定和分化是必不可少的。总体目标是确定 SfrslO在视网膜发育过程中的表达、功能及靶点我采用了原位杂交技术， 免疫荧光和微阵列分析确定SfrslO在视网膜发育过程中的表达。 总之，SfrslO似乎在祖细胞和分化中的无长突细胞中表达。有趣的是， 从单个视网膜细胞获得的微阵列数据显示，某些祖细胞标志物，如Cycling DL 和Sfrsp2随着SfrslO的表达而丰富。为了确定SfrslO的功能，！有 利用体内电穿孔技术将F^NaI或错义表达载体导入PO视网膜前体细胞。1 他们发现，SfrslO功能的丧失会导致Mullerglia数量的增加，而损害神经元的数量。 这项实验利用哈密尔顿针将质粒送入视网膜下间隙。不过，这个 这种方法经常会对视网膜造成损害。我现在采用了一种不同的方法，用玻璃 在视网膜上留下微不足道印记的针头。而在赠款中报告的RNAi构建已经 用于确定SfrslO功能的条件性基因敲除小鼠对这一提议至关重要。这个 康涅狄格大学已经开始生产这样的鼠标，我将在那里开始我的职位 2010年8月1日，一位助理教授。当我在我的实验室里继续这个项目的时候 康涅狄格州，我将进一步细化SfrslO的表达式，用 电穿孔技术。最后，有条件基因敲除的小鼠将与视网膜特定的CRE系杂交 其次对其表型进行了详细分析。此外，基因敲除的小鼠视网膜细胞将用于深部 (454)查封以识别SfrslO的目标。我实验室这项工作的扩展将为我提供 我打算用来获得ROL基金的初步数据，这将开启我在科学领域的独立事业。