title: Affinity-based isolation of tagged nuclei from Drosophila tissues for gene expression analysis.
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.
authors.affiliation: Department of Biochemistry, Purdue University.
authors.forename: Jingqun
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Biochemistry, Purdue University; vweake@purdue.edu.
authors.forename: Vikki Marie
authors.initials: VM
authors.lastname: Weake
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004331:Drosophila melanogaster; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017434:Protein Structure, Tertiary
chemical_list: D009687:Nuclear Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins