Tann Jason Y, Xu Fangke, Kimura Minami, Moore Adrian W
Laboratory for Neurodiversity, RIKEN Center for Brain Science, Wako-shi, 351-0106, Japan.
Laboratory for Neurodiversity, RIKEN Center for Brain Science, Wako-shi, 351-0106, Japan
Cold Spring Harb Protoc. 2024 Dec 2;2024(12):pdb.prot108147. doi: 10.1101/pdb.prot108147.
Mosaic analysis with a repressible cell marker (MARCM) is used in research to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations. Previous protocols that outlined experimental procedures to create MARCM clones in da neurons used a heat shock promoter to drive Flippase (FLP) expression; such an approach requires laborious embryo collection and heat shock steps, and it creates clones in other tissues besides the da neurons. The updated protocol described here outlines the use of FLP expression driven by a sensory organ precursor promoter (SOP-FLP); it requires no embryo collection or manipulation steps and creates clones exclusively in the peripheral sensory neuron lineage.
利用可抑制细胞标记的镶嵌分析（MARCM）在研究中用于在其他方面为杂合子的果蝇中创建标记的纯合子突变细胞克隆。它允许研究胚胎致死基因的作用以及确定突变表型的细胞自主性。当与靶向质膜的荧光蛋白一起用于树突状分支（da）神经元时，MARCM允许鉴定纯合子突变神经元，并在活体制备和固定体制备中对树突分支进行清晰成像。以前概述在da神经元中创建MARCM克隆的实验程序的方案使用热休克启动子来驱动翻转酶（FLP）表达；这种方法需要费力的胚胎收集和热休克步骤，并且它会在da神经元之外的其他组织中创建克隆。此处描述的更新方案概述了由感觉器官前体启动子（SOP-FLP）驱动的FLP表达的使用；它不需要胚胎收集或操作步骤，并且仅在外周感觉神经元谱系中创建克隆。
