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首页> 外文期刊>Genomics >Functional screening for proapoptotic genes by reverse transfection cell array technology.
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Functional screening for proapoptotic genes by reverse transfection cell array technology.

机译：通过逆转染细胞阵列技术对促凋亡基因进行功能筛选。

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摘要

Application of mathematical algorithms to sequenced whole genomes revealed a large number of predicted genes, requiring functional assays for their characterization in a high-throughput manner. Here, we report on the development of a screening assay, which is based on reverse transfection of cellular arrays and subsequent analysis of cell morphology to identify novel proapoptotic genes. Expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pEYFP to screen for apoptotic body formation, based on EYFP fluorescence. The assay was validated and applied to 382 human sequence-verified full-length open reading frames, most of them of unknown function. In this initial screening, proapoptotic effects could be demonstrated for 10 of these genes. For 6 of them apoptosis induction could be confirmed both by TUNEL assay and by FACS analysis of cells stained according to Nicoletti: 1 gene was not yet annotated for an apoptotic function (ST6GAL2), while 5 genes were without annotated function (FLJ20551, CXorf12, FAM105A, TMEM66, C19orf4). Our study demonstrates the potential of this method to characterize functionally genes of unknown function in a highly parallel format.
机译：将数学算法应用于已测序的全基因组序列揭示了大量预测基因，需要以高通量方式对其特征进行功能分析。在这里，我们报告了筛选测定法的发展，该测定法是基于细胞阵列的反向转染和随后的细胞形态分析以鉴定新的促凋亡基因。将含有全长cDNA的表达质粒与报道质粒pEYFP共转染，以基于EYFP荧光筛选凋亡小体的形成。该检测方法得到验证，并应用于382个经过人序列验证的全长开放阅读框，其中大多数功能未知。在此初始筛选中，可以证明其中10个基因具有促凋亡作用。对于其中6个细胞，可以通过TUNEL分析和通过Nicoletti染色的细胞的FACS分析来确认凋亡诱导：1个基因尚未标注凋亡功能（ST6GAL2），而5个基因没有标注凋亡功能（FLJ20551，CXorf12， FAM105A，TMEM66，C19orf4）。我们的研究证明了这种方法以高度平行的形式表征功能未知的功能基因的潜力。

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