The Role of Alternative Splicing Factor Sfrs10 in Neural Development

选择性剪接使赫特人能够从相对较少的（约24，000种）蛋白质中产生大量的蛋白质组多样性 编码基因这个ROO提案是深入了解选择性剪接的错误的总体目标的一部分 在视网膜发育中。1人选择以SfrslO为重点来解决这个问题， 假设它是细胞命运决定和分化所必需的。总体目标是确定 SfrslO在视网膜发育过程中的表达、功能和靶标。我采用了原位杂交技术， 免疫荧光和微阵列分析以确定视网膜发育期间SfrslO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，某些祖细胞标记物如循环D1 和Sfrsp 2的表达沿着Sfrs 10的表达而富集。为了确定SfrslO的功能，！有 采用体内电穿孔将F1 NAi或错表达构建体递送到PO视网膜祖细胞中。1 已经发现Sfr 10功能的丧失导致以神经元为代价的Mullerglia数量的增加。 该实验利用汉密尔顿针将质粒递送到视网膜下腔中。但这 这种方法经常会对视网膜造成损伤。1现在采用了一种不同的方法， 在视网膜上留下可忽略不计的印记。虽然在赠款中报道的RNAi构建体已经被证实是有效的， 用于确定Sfrs 10的功能，条件性敲除小鼠对于该提议至关重要。的 这种鼠标的生产已经在康涅狄格大学开始，在那里我将开始我的职位， 2010年8月1日成为助理教授当我在纽约大学的实验室里继续这个项目时， 康涅狄格州，我将进一步细化SfrslO的表达，执行SfrslO函数的增益和损失， 电穿孔技术最后，将条件敲除小鼠与视网膜特异性Cre系杂交， 然后详细分析表型。此外，敲除小鼠视网膜细胞将用于深 (454)测序以鉴定SfrslO的靶标。在我的实验室里扩展这项工作将为我提供 初步数据，我打算使用的研究生奖学金，应该启动我的独立职业生涯的科学。

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.