Genetics
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使用果蝇的异色素促进药物的鉴定筛选方法
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Summary March 12th, 2020
Please note that all translations are automatically generated.
Click here for the English version.
果蝇是一种广泛使用的实验模型，适用于筛选具有癌症治疗潜在应用的药物。在这里，我们描述了使用果蝇变眼色表型作为筛选小分子化合物，促进异构素形成的方法。
Transcript
目前，没有药物在促进异色素形成方面已建立良好。在这里，我们提出了一个简单而有效的方法来识别促进这一过程的小分子化合物。我们的筛选协议提供了一个简单，廉价，易于遵循体内的异色素促进药物鉴定系统。
识别促进异色素形成的化合物可能导致发现治疗和癌症治疗的癌症机制。这种成本效益已经测量用于识别嗜血杆菌中的异色素促进化合物也可用于计算形式命中，因为它是一个预防措施，以最高的细胞池细胞筛选。我们的筛选协议也可用于快速引导屏幕，因为这个果蝇TranSheet系统产生一个变异的眼睛颜色表型，与苍蝇中存在的异色素量成反比。
帮助演示这个程序的将是肯尼道，一个本科生从我们的实验室。在使用适当的溶剂和所需的浓度确定并准备药物库进行筛选后，在 25 由 95 毫米食物小瓶中与三只或三只（三只处女雌蝇）交叉三只雄蝇。与九毫米的标准果蝇食品介质。
为每种药物设置三个复制小瓶。为屏幕添加一个控制小瓶。允许苍蝇在室温下产卵两天。
在十字架的第二天，用短暂的二氧化碳来麻醉苍蝇。将父母用含有70%乙醇的瓶子里丢弃。接下来，稀释每个药物化合物从初始库存参加微摩尔最终浓度在33%DMSO在水中。
并添加60微升的药物，或控制33%DMSO单独，在飞的食物的顶部。然后确认小瓶中不存在母蝇。并将药物溶液添加到适当的小瓶中。
F1苍蝇出现两天后，一次从一个小瓶将麻醉苍蝇转移到一个多孔解剖垫上，二氧化碳从滤纸下面过滤通过。在解剖显微镜下，检查每只苍蝇，通过缺乏卷曲的 O 显性卷曲翅膀表型来识别所有异质雄性。在一到五的尺度上给每个异质雄性的眼睛颜色评分。
将一个分数分配给小于 5% 的红色散射眼总表面积。向具有 6 到 25% 红斑眼总表面积的苍蝇添加两个分数。一个三飞与26至50%红斑眼总表面积。
一个四飞与51至75%红斑眼总表面积。和五只苍蝇的红斑眼总表面积大于75%。然后计算每个小瓶中按三倍得分的所有雄性的所有男性的均色指数。
为了确认这些化合物确实促进了异色素的形成，可以执行一种不同的验证技术，如西方印迹。在这个具有代表性的实验中，一个小分子药物库，肿瘤学集3，它由97个FDA授权的肿瘤药物组成，使用DX1菌株的果蝇黑色素进行筛选。根据筛选测定，在库中编码为E7的二氨蝶酯在Dx1菌株中引起最大的变化。
暗示明氨蝶酯可能是未来癌症靶向治疗最有希望的促进异色素的药物。为了进一步确认该化合物促进异色素转化，进行了西部印迹数据。演示异铬素格式相关蛋白的上规，H3K9三甲基化，并验证筛选结果。
关键步骤是根据预定的研究路线，按一到五的尺度对每只白色11-18 DX1异质雄性苍蝇的眼睛颜色进行评分。我们可以用西方的污点或免疫污迹来确认命中。发现疑似促进异色素形成的化合物，也可能导致发现癌症发育的IV遗传机制，并在人类患者中进行临床试验。
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