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

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 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：解析光感受器细胞分化过程中的基因调控网络。 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细胞的基因表达谱。 研究背景：视杆光感受器细胞的发生在视网膜发育过程中具有宽泛的时间窗口，最早始于胚胎日12.5（E12.5），并在出生后0-2天（P0-P2）达到峰值。在胚胎日16.5（E16.5）时，已有少量早期生成的视杆细胞，但视杆细胞发生的峰值尚未到来；出生后2天（P2）时，绝大多数视杆光感受器细胞已生成。出生后6天（P6），视杆细胞特异性结构/功能基因开始表达；出生后10天（P10），外节形态发生过程启动。至4周龄时，视网膜发育完全并具备生理功能。神经视网膜亮氨酸拉链（Nrl）是视杆细胞特异性转录因子，亦是视杆光感受器细胞最早的标记物之一。Nrl启动子可在视杆光感受器细胞退出细胞周期后不久，仅驱动其表达绿色荧光蛋白（Green Fluorescent Protein, GFP）。在Nrl敲除的遗传背景中，绿色荧光蛋白的表达可在S视蛋白阳性细胞中被检测到，这表明在Nrl缺失的情况下，细胞命运发生了从视杆细胞到视锥细胞的转化。 实验设计：从野生型GFP（WT-Gfp）或Nrl敲除型GFP（Nrl-knockout-Gfp）视网膜中分离的GFP阳性光感受器细胞，通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）在5个明确的发育阶段（E16、P2、P6、P10及4周龄）进行富集纯化。使用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小鼠全基因组表达芯片430 2.0上进行杂交，随后按照标准流程进行扫描。每个时间点均设置4次生物学重复。 研究结论：通过对比不同发育阶段的基因表达谱，可获得关于光感受器细胞分化背后分子事件的全新认知。 关键词：转录因子，发育，光感受器细胞，视网膜，神经元，分化，基因调控，基因芯片，基因表达谱分析，细胞类型对比 在野生型背景的小鼠（Wt-Gfp小鼠）中，处于有丝分裂后状态的视杆前体细胞与成熟视杆光感受器细胞可在Nrl启动子的调控下被GFP标记。当将该品系与Nrl敲除背景小鼠杂交后，得到的Nrl-ko-Gfp小鼠中，转化型S视锥细胞可被GFP标记。从Wt-Gfp或Nrl-ko-Gfp视网膜中分离的GFP阳性光感受器细胞，通过FACS在5个明确的发育阶段（E16、P2、P6、P10及4周龄）进行富集纯化。使用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小鼠全基因组表达芯片430 2.0上进行杂交，随后按照标准流程进行扫描。每个时间点均设置4次生物学重复。