Universität zu Köln

Mundorf, Juliane and Uhlirova, Mirka ORCID: 0000-0002-5735-8287 (2016). The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics. J. Vis. Exp. (116). CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS. ISSN 1940-087X

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Abstract

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome- wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Mundorf, Juliane UNSPECIFIED UNSPECIFIED UNSPECIFIED Uhlirova, Mirka UNSPECIFIED orcid.org/0000-0002-5735-8287 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-261677
DOI:	10.3791/54585
Journal or Publication Title:	J. Vis. Exp.
Number:	116
Date:	2016
Publisher:	JOURNAL OF VISUALIZED EXPERIMENTS
Place of Publication:	CAMBRIDGE
ISSN:	1940-087X
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CELL MARKER MARCM; IMMUNE-RESPONSE; ONCOGENIC RAS; INVASION; METASTASIS; MALIGNANCY; POLARITY; PROTEIN; SYSTEM; FOS Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26167