alexa Part II. Fly-FISHing: A Protocol to Localize Single Copy Genes inside the Nucleus in Whole Mount Embryos.
Medicine

Medicine

Alternative & Integrative Medicine

Author(s): Authors Gemkow MJ

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Excerpt The nucleus is the place where transcription factors, DNA and RNA polymerases, fulfil their tasks. The chromatin is made up of an undefined number of proteins that all contribute to the way the information contained in the genome is utilized. How the nucleus is organized and how all of the processes are interwoven inside the nucleus is an open and ongoing area of debate (the following books give a good overview of this field: Chromatin: Structure & Function, Alan Wolffe, 1998; Chromatin Structure and Gene Expression, Ed. Sarah C. R. Elgin, 1995; CSH Symposia on Quantitative Biology, Vol. 58, 1993). Antibody staining to proteins involved in mitosis and transcription revealed complex and dynamic patterns (Buchenau 1993a, 1993b, 1997). Drosophila offers a unique system to study changes in nuclear architecture and to reveal the dynamics of these processes. This can be done by injection of fluorescently labeled antibodies into Drosophila embryos or by using the GFP technology for in vivo studies (Amsterdam et al. 1995; Davis et al. 1995; Yeh et al. 1995). Alternatively, fixed embryonic material can be extremely helpful for visualizing dynamics in changing structures because in each set of experiments, different developmental timepoints are represented that can be sorted to reflect the developmental progression. In this technical note, I describe a fluorescence in situ technique that can be used to localize single copy genes in three dimensions inside the nucleus. This technique was successfully used to determine the pairing kinetics of the Bithorax-complex (BX-C) during embryogenesis and to gain insights into the mechanism of transvection (Gemkow et al., 1998 1996). Localization and pairing studies for chromosomal loci on the second chromosome were also performed (Hiraoka et al. 1993; Fung et al. 1998 and references therein). The protocol given here can also be used in combination with antibody staining inside the nucleus, which enabled us to colocalize the BX-C with proteins of the Polycomb-group. For this purpose, a regular antibody protocol has to be performed after hybridization. An alternative protocol from Gunawardena and Rykowski (1994) can be found in: Drosophila melanogaster: Practical Uses in Cell and Molecular Biology; edited by Goldstein and Fyrberg (Methods in Cell Biology, Vol. 44). Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM).
This article was published in Part II and referenced in Alternative & Integrative Medicine

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