Lee Hyung-Kook Peter, Wright Ashley P, Zinn Kai
Division of Biology, California Institute of Technology, USA.
J Vis Exp. 2009 Dec 29(34):1647. doi: 10.3791/1647.
Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.
处于胚胎发育14至17阶段的果蝇胚胎可以很容易地进行解剖,以制作“去壳”标本。在这些标本中,中枢神经系统位于中间,两侧是体壁。许多不同的表型已通过此类标本进行了检测。在大多数情况下,这些去壳标本是通过解剖抗体染色的固定整装胚胎制作而成的。然而,这些“固定解剖”存在一些缺点。它们执行起来耗时,而且很难从通过绿色荧光蛋白(GFP)平衡染色体维持突变的品系中筛选出突变体(GFP阴性)胚胎。自2002年以来,我们团队一直在进行缺失和异位表达筛选,以鉴定孤儿受体的配体。为了做到这一点,我们开发了简化的方案,用于对包含数百个平衡品系的样本进行活胚胎解剖和抗体染色。我们得出的结论是,通过活解剖检查大量品系样本中的表型比通过固定解剖效率要高得多。使用此处描述的方案,一名经过培训的人员每天最多可以筛选10个品系的表型,在复式显微镜下检查每个品系的4 - 7个突变胚胎。这使得能够鉴定出赋予细微、低 penetrance 表型的突变,因为使用40倍水浸物镜在高倍放大下,每个品系最多可对70个半体节进行评分。