Drosophila embryo extract preparation

The preparation of extracts from preblastoderm Drosophila embryos and their use for assembling chromatin has been described in detail by Becker et al. (1994). In this section I briefly outline these procedures as well as the in vitro conditions under which nucleosome remodeling occurs. 

Preparation of Nuclear Extracts from Drosophila Embryos and In Vitro Transcription Analysis ... 

The production of a high-quahty Drosophila embryonic cytoplasmic extract for use in protein purification or biochemistry is relatively easy, providing population cages are available that produce at least 5 g of embryos during a 3-hour period (for methods to maintain population cages, see Sisson, this volume). Smaller quantities of embryos can be used to produce extracts that are useftil for in vitro biochemical assays (see, e.g., Moritz et al. 1998), and thus it should be possible to make extracts from mutant stocks that could then be tested in vitro. Protocol 33.1 describes the collection and dechorionation of embryos for making cytoplasmic extracts. It is fairly easy to prepare even very concentrated cytoplasmic extracts from Drosophila embryos, with protein concentrations of 50-75 mg/ml (see Protocol 33.2). 

Extracts from Drosophila melanogaster (Oregon R, P2 strain) embryos can be prepared in the following way (Berrios and Avilion, 1990 Kawasaki et ai,... 

In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

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