XB-ART-46513Nat Protoc. February 1, 2013; 8 (2): 310-24.
A single Drosophila embryo extract for the study of mitosis ex vivo.
Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
PubMed ID: 23329004
Article link: Nat Protoc.
Grant support: Cancer Research UK, Cancer Research UK