Murphy Timothy P, Luu Dan D, DeSimone Jessica A, O'Brien Thomas C, Lally Christopher J, Lindblad Jillian J, Webster Sarah M
Department of Biology, College of the Holy Cross; School of Medicine, Georgetown University.
Department of Biology, College of the Holy Cross.
J Vis Exp. 2015 Dec 30(106):e53537. doi: 10.3791/53537.
Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.
由于与哺乳动物内耳毛细胞存在结构和功能同源性,支配果蝇外周感觉器官的神经元为机械感觉研究提供了一个出色的模型系统。本方案描述了果蝇的一种简单触觉行为,可用于鉴定干扰机械感觉的突变。对果蝇胸部的刚毛进行触觉刺激会引发第一或第三条腿的梳理反射。干扰机械转导(如NOMPC)或反射弧其他方面的突变可抑制梳理反应。传统的成年行为筛选会遗漏在发育过程中起重要作用的突变体。相反,本方案将触摸屏与带有可抑制细胞标记的镶嵌分析(MARCM)相结合,仅允许产生有限区域的纯合突变细胞,并通过绿色荧光蛋白(GFP)的表达进行标记。通过测试MARCM克隆的异常行为反应,有可能筛选致死性P因子突变体集合,以寻找更多传统方法会遗漏的参与机械感觉的新基因。