A manual small molecule screen approaching high-throughput using zebrafish embryos.
A manual small molecule screen approaching high-throughput using zebrafish embryos.
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DOI：
10.3791/52063
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发表时间：
2014-11-08
期刊：
影响因子：
--
通讯作者：
Wingert RA
中科院分区：
文献类型：
--
作者：
Poureetezadi SJ;Donahue EK;Wingert RA
斑马鱼由于与人类具有相当程度的遗传保守性，已成为广泛用于研究发育生物学机制和研究人类疾病病理学的模式生物。化学遗传学需要测试小分子对生物过程的影响，并且正在成为一种流行的识别治疗化合物的转化研究方法。斑马鱼特别吸引人用于化学遗传学，因为它们能够产生大量透明的胚胎，这些胚胎是外部受精的。此外，斑马鱼胚胎可以很容易地通过向胚胎培养基中简单添加化合物进行药物处理。利用全载原位杂交技术（WISH），可以清楚地看到斑马鱼胚胎中mRNA的表达。同时，利用化学遗传学和WISH，斑马鱼成为确定小分子的细胞和生理效应的强有力的整个生物体环境。在利用基于机器的筛选程序的技术方面取得了创新进展，但是对于许多实验室来说，这种选择无法获得或仍然成本过高。这里描述的协议解释了如何执行手动高通量化学遗传筛选，这需要基本的资源，可以由一个人或小团队在有效的时间内完成。因此，该协议提供了一种可行的策略，可以由研究小组在斑马鱼中执行化学遗传学，这对于获得对发育过程，疾病机制的基本见解以及识别具有医学相关应用的新化合物和信号通路是有用的。
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
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