荧光激活细胞分选转基因标记斑马鱼神经元

Fluorescence-activated Cell Sorting(FACS)of Fluorescently Tagged Cells from Transgenic Zebrafish Larvae for Gene Expression Analysis

对常规试验方法加以改进,使之适用于斑马鱼脑部荧光标记神经细胞的分选。以lhx5∶Kaede转基因株系为例,利用转基因斑马鱼脑部制备单细胞悬液进行荧光激活细胞分选(Fluorescence-activated cell sorting,FACS),获得lhx5荧光标记细胞。提取总RNA后进行基因芯片分析从而了解lhx5标记神经元的基因表达特性。结果显示,通过本流程可成功制备斑马鱼幼鱼脑部单细胞悬液,lhx5∶Kaede平均荧光阳性率为6%。提取RNA质量及总量均能满足基因芯片要求。基因芯片结果显示lhx5及其他标记分子的表达均得到了显著富集。有效地分离斑马鱼幼鱼中枢神经系统荧光标记细胞,可用于特定细胞群的基因表达谱分析。

It was to optimize commonly used FACS protocols to apply to fluorescence-labeled neurons of zebrafish, lhx5 ： Kaede transgene line as an example was used to describe a successful protocol for isolating and expression-profiling live fluorescent-protein-labeled neurons from transgenic zebrafish brains. Numerous information of ltrx5 labehng ceils can be obtained by RNA profiling and would contribute to further studies. Results showed that on average, 6% of the single cell suspensions was keade-positive. The RNA extracted from selected cells was sufficient quality and quantity for microarray analysis. Microarray data showed the enrichment of both lhx5 and other molecular makers. It proved that this protocol for dissociating and sorting fluorescent-labeled neurons from central nervous system of zebrafish larve, is applicable for gene expression profiling of the target cell groups.