ISSN 1006-298X      CN 32-1425/R

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肾脏病与透析肾移植杂志 ›› 2014, Vol. 23 ›› Issue (3): 201-206.

• 论文 •    下一篇

利用荧光激活细胞分选技术获取荧光蛋白标记肾小球足细胞

  

  • 出版日期:2014-06-28 发布日期:2014-07-02

Using Fluorescence-activated cell sorting isolate  glomerular podocyte in double fluorescent reporter mice

  • Online:2014-06-28 Published:2014-07-02

摘要:

目的:本实验旨在以双荧光蛋白标记转基因小鼠为动物模型,建立适用于小鼠荧光标记足细胞的分选方法。 方法:对现有常规实验方法加以优化,在使用报告基因小鼠的前提下辅以更有效的磁珠灌注法提取肾小球,进一步制备单细胞悬液进行荧光激活细胞分选(FACS),获得绿色荧光蛋白标记足细胞。利用荧光显微镜及荧光定量PCR分析证实分选所得足细胞基因表达特性。 结果 通过本试验方法可更有效制备肾小球单细胞悬液和高纯度的足细胞,每只小鼠可提取超过3×105的足细胞,占细胞总量的14.93%,质量可满足后续转录组、蛋白组学等相关研究。结论 应用荧光激活细胞分选技术可有效的从荧光标记小鼠中提取高纯度足细胞,为足细胞基因组学、蛋白组学水平的研究奠定基础。

关键词: 荧光激活细胞分选, 报告基因小鼠, 足细胞

Abstract:

Abstract Objective To describe a double fluorescent reporter mouse model to establish optimized protocol for GFP labeled podocyte isolation. Methodology: Based on fluorescent reporter transgenic mouse model together with modified extraction protocol, we demonstrated stable method to isolate podocyte using Fluorescence-activated cell sorting(FACS). Result:The efficient single cell dissociation to yield more than 300,000 purified podocytes per mouse were obtained allowing for global, unbiased downstream applications. Efficiency and purity could be monitored by evaluating sorted cells under a fluorescence microscope. Conclusion: RT-PCR analysis revealed that podocyte specific gene were highly enriched in GFP+ fraction, which was sufficient for further transcriptome profiling as well as quantitative proteomic analysis in omics approach.

Key words: FACS, reporter mice, podocyte