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首页> 外文期刊>Experimental Eye Research >Isolation of photoreceptors from mature, developing, and regenerated zebrafish retinas, and of microglia/macrophages from regenerating zebrafish retinas
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Isolation of photoreceptors from mature, developing, and regenerated zebrafish retinas, and of microglia/macrophages from regenerating zebrafish retinas

机译：从成熟，开发和再生斑马鱼视网膜和从再生斑马鱼视网膜的微胶质/巨噬细胞分离感光体

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This paper describes experimental procedures for the dissociation of retinal cells of the zebrafish (Dania rerio) for subsequent fluorescence-activated cell sorting (FACS) and gene expression studies. Methods for dissociation of zebrafish retinas followed by FACS and RNA isolation were optimized. This methodology was applied to isolate pure sorted samples of rods, long wavelength-sensitive (LWS) cones, medium wavelength-sensitive (MWS; RH2-2) cones, short wavelength-sensitive (SWS2) cones, and UV-sensitive (SWS1) cones from retinas obtained at selective life-history stages of the zebrafish, and for some of these photoreceptors, following retinal regeneration. We also successfully separated lws1-expressing and lws2-expressing LWS cones from fish of a transgenic line in which lws1 is reported with green fluorescence protein (GFP) and lws2 is reported with red fluorescence protein (RFP). Microglia/macrophages were successfully sorted from regenerating retinas (7 days after a cytotoxic lesion) of a transgenic line in which these immune cells express GFP. Electropherograms verified downstream isolation of high-quality RNA from sorted samples. Examples of post-sorting analysis, as well as results of qRTPCR studies, validated the purity of sorted populations. For example, qRT-PCR samples derived from isolated Rh2-2 cones contained detectable rh2-2 (opn1mw2) opsin transcripts, but iws opsin transcripts (lws1/opn1lw1, lws2/opn1lw2) were not detected, suggesting that the procedure likely separated double cone pairs. Through this method, pure, sorted cell samples can provide RNA that is reliable for downstream gene expression analyses, such as qRT-PCR and RNA-seq, which may reveal molecular signatures of photoreceptors and microglia for comparative transcriptomics studies.
机译：本文介绍了用于斑马鱼（Dania RERIO）视网膜细胞的实验程序，用于随后的荧光活性细胞分选（FACS）和基因表达研究。优化了对斑马鱼视网膜解离的方法，然后进行FAC和RNA分离。该方法应用于分离棒的杆，长波长敏感（LWS）锥体，中波长敏感（MWS; RH2-2）锥体，短波长敏感（SWS2）锥体和紫外敏感（SWS1）在斑马鱼的选择性生命历史阶段获得的视网膜，以及这些感光体之后的一些视网膜再生。我们还成功地将LWS1表达和LWS2表达的LWS锥体从转基因系列中分离，其中LWS1与绿色荧光蛋白（GFP）和LWS2报告，具有红色荧光蛋白（RFP）。通过这些免疫细胞表达GFP的转基因系的再生视网膜（在细胞毒性病变后7天后，成功地分类了微胶质细胞/巨噬细胞。电泳图验证了从分选样品中验证了高质量RNA的下游分离。分类后分析的实例，以及QRTPCR研究的结果，验证了分类种群的纯度。例如，衍生自分离的RH2-2锥体的QRT-PCR样品含有可检测的RH2-2（OPN1MW2）OPSIN转录物，但没有检测到IWS OPSIN转录物（LWS1 / OPN1LW1，LWS2 / OPN1LW2），表明该过程可能分离双锥体对。通过该方法，纯的分选细胞样本可以提供可靠的下游基因表达分析的RNA，例如QRT-PCR和RNA-SEQ，这可能揭示感光体和微胶质细胞的分子签名进行比较转录组研究。

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