Targeting of GFP to new-born rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Mus musculus

Purpose: To investigate the gene regulatory networks during photoreceptor differentiation. Special aims: To generate gene expression profiles of purified photoreceptors at distinct developmental stages and from different genetic backgrounds. Background: Rod photoreceptor genesis spans a broad temporal window during retinal development. It starts as early as E12.5 and peaks at P0-P2. At E16.5, there are some early born rods but the peak of rod genesis does not occur. At P2, the majority of rod photoreceptors are born. At P6, rod specific structural/functional genes begin to express. At P10, Outer segments morphogenesis is taking place. At 4 weeks, retinal development is complete and retina is functional. Nrl is a rod specific transcription factor and one of the earliest markers of rod photoreceptors. Nrl promoter drives the expression of GFP exclusively to rod photoreceptors shortly after they exit cell cycle. In the Nrl-knockout background, the expression of GFP is detected in S-opsin positive cells, which suggested a cell fate transformation from rods to cones in the absence of Nrl. Design: GFP positive photoreceptors from the WT-Gfp or Nrl-knockout-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol reagent. Around 50 ng of total RNA was used for linear amplification and biotin labeling followed Nugen kit protocol. Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point. Conclusion: By comparing the gene expression profiles from different developmental stages, we can obtain novel insights into molecular events underlying photoreceptor differentiation. Keywords: Transcription factor, development, photoreceptor, retina, neuron, differentiation, gene regulation, microarray, gene profiling, cell type comparison Overall design: Postmitotic rod precursors and mature rod photoreceptors are tagged by GFP under the control of an Nrl promoter in the wild type background (Wt-Gfp mice). When cross-bred into the Nrl-knockout background (Nrl-ko-Gfp mice), the transformed “S-cones” are tagged by GFP. GFP positive photoreceptors from the Wt-Gfp or Nrl-ko-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol regent and around 50 ng of total RNA was used for linear amplification and biotin labeling following Nugene kit protocol. Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.

研究目的：探究光感受器分化过程中的基因调控网络。
具体研究目标：获取不同发育阶段、不同遗传背景下纯化光感受器的基因表达谱。
研究背景：视网膜发育过程中，杆状光感受器（rod photoreceptor）的发生具有较宽的时间窗口：最早始于胚胎期12.5天（E12.5），在出生后0-2天（P0-P2）达到峰值。E16.5时已有少量早期生成的杆状细胞，但杆状细胞发生的峰值尚未出现；P2时，绝大多数杆状光感受器生成；P6时，杆状细胞特异性结构/功能基因开始表达；P10时，外节（Outer segments）发生形态发生；4周时，视网膜发育完成并具备功能。Nrl是杆状光感受器特异性转录因子，也是该细胞类型最早的标志物之一。Nrl启动子可在杆状光感受器退出细胞周期后不久，仅驱动其表达绿色荧光蛋白（GFP）。在Nrl基因敲除（Nrl-knockout）背景中，GFP可在S-视蛋白（S-opsin）阳性细胞中被检测到，这表明缺失Nrl时，细胞命运会从杆状细胞向锥状细胞转化。
实验设计：从野生型GFP标记（WT-Gfp）或Nrl基因敲除GFP标记（Nrl-knockout-Gfp）的视网膜中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）在5个明确的发育阶段（E16、P2、P6、P10及4周）富集纯化GFP阳性光感受器。采用Trizol试剂提取总RNA，取约50 ng总RNA按照Nugen试剂盒流程进行线性扩增与生物素标记。将片段化的cDNA在Affymetrix小鼠基因组表达芯片430 2.0上进行杂交，随后按照标准流程进行扫描。每个时间点设置4次生物学重复。
研究结论：通过比较不同发育阶段的基因表达谱，可获得光感受器分化背后分子事件的全新认知。
关键词：转录因子（Transcription factor）、发育、光感受器、视网膜、神经元、分化、基因调控、微阵列（microarray）、基因表达谱分析、细胞类型比较
整体实验设计：在野生型背景（Wt-Gfp小鼠）中，Nrl启动子调控的GFP可标记有丝分裂后杆状前体细胞与成熟杆状光感受器。将该品系与Nrl基因敲除背景（Nrl-ko-Gfp小鼠）回交后，转化得到的“S型锥状细胞”可被GFP标记。从Wt-Gfp或Nrl-ko-Gfp视网膜中分离的GFP阳性光感受器，通过FACS在E16、P2、P6、P10及4周这5个发育阶段完成富集纯化。采用Trizol试剂提取总RNA，取约50 ng总RNA按照Nugene试剂盒流程进行线性扩增与生物素标记。将片段化的cDNA在Affymetrix小鼠基因组表达芯片430 2.0上杂交，随后以标准流程扫描。每个时间点均设置4次重复。