NDLI: Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Content Provider	PubMed Central
Author	Ma, Jingqun Marie, Weake Vikki
Copyright Year	2014
Abstract	Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.
Related Links	http://dx.doi.org/10.3791/51418
Starting Page	51418
File Format	PDF
ISSN	1940087X
e-ISSN	1940087X
Journal	Journal of Visualized Experiments : JoVE
Issue Number	85
Language	English
Publisher	MyJove Corporation
Publisher Date	2014-03-01
Access Restriction	Open
Rights Holder	MyJove Corporation
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Immunology and Microbiology(all) Chemical Engineering(all) Neuroscience(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Neuroscience Immunology and Microbiology Biochemistry, Genetics and Molecular Biology Chemical Engineering

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