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

Summary

Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

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.

Introduction

Drosophila tissues such as the central nervous system contain a complex mixture of cell types. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it is first necessary to isolate a homogenous population of specific cells in sufficient quantities to enable downstream applications. Methods to isolate cells from intact tissues include laser microdissection, and fluorescence-activated cell sorting (FACS) of whole cells. While FACS has been used to isolate cells and nuclei from Drosophila embryos and from Caenorhabditis elegans for gene expression and chromatin profiling^1-3, FACS and lase....

Protocol

1. Generation and Characterization of EGFP::KASH Drosophila

1. Cross Gal4 driver flies to UAS-EGFP::Msp-300^KASH flies. Alternatively, recombinant flies that carry both the Gal4 driver and the UAS-EGFP::Msp-300^KASH transgene can be generated using standard genetic techniques. This second approach is advantageous if large numbers of progeny are required.
2. Characterize expression of the EGFP::KASH marker using standard microscopy techniques. Note: EGFP expression can be observed by standard microscopy techniques in dissected, fixed Drosophila tissues, or in whole larvae, and does....

Results

The repo-GAL4 driver (Bloomington stock number 7415) is specifically expressed in glial cells in the Drosophila nervous system at multiple stages of development¹⁸. Flies were generated that stably express UAS-EGFP::Msp-300^KASH under control of the repo-GAL4 driver using standard genetic techniques. To characterize the expression pattern of the EGFP::KASH transgene in these flies, the pattern of GFP expression in the dissected optic lobe and eye imaginal disc from .......

Discussion

This protocol can be used to isolate or highly enrich specifically tagged nuclei from mixed cell populations in Drosophila embryonic or larval tissues. Using this protocol, nuclei can be isolated from dissected tissues in approximately 1 hr. The purity and yield of the isolated nuclei must be experimentally determined for each cell type. It can be difficult to quantify the bead-bound nuclei at the post-isolation stage of the protocol using a hemocytometer because of the beads present in the sample. Thus, if exac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-chaoptin antibody (mouse, monoclonal)	Developmental Studies Hybridoma Bank	mAB24B10
Anti-GFP antibody (mouse, monoclonal)	Roche	11814460001	This specific antibody is recommended for this protocol. However, alternative antibodies could also be used.
2x Bullseye EvaGreen qPCR Mastermix with ROX	MidSci	BEQPCR-R
DAPI (4',6-diamidino-2-phenylindole, dihydrochloride salt)	Biotium	40011
Dounce Homogenizer (1 ml)	VWR	62400-595
Dumont #5 Tweezers, 11 cm	World Precision Instruments	14095
Dynabeads Protein G for Immunoprecipitation	Invitrogen	10003D	This specific brand of magnetic beads is recommended for this protocol.
EpiScript Reverse Transcriptase	Epicentre	ERT12910K	http://www.epibio.com/docs/default-source/protocols/episcript-reverse-transcriptase.pdf
Falcon cell strainers, 40 μm pore size	VWR	21008-949	Alternative pore sizes could be used, depending on the size of the target cell nuclei.
Magnetic stand (8 x 1.5 ml tubes)	Millipore	LSKMAGS08
Mini tube rotator	Genemate	H-6700
Qubit starter kit	Life Technologies	Q32871	Similar fluorescence-based methods for quantifying RNA yield could be used. http://tools.invitrogen.com/content/sfs/manuals/mp32852.pdfZ
RNAsecure (Ambion)	Life Technologies	AM7010	The use of this reagent to inactivate RNAses is optional.
RT-PCR machine Thermal Cycler	BioRad	CFX Connect Thermal Cycler
Siliconized 9-well glass plate	Hampton Research	HR3-134
Stereomicroscope Fluorescence Adapter (Royal Blue (440-460 nm) excitation + yellow barrier	Nightsea		This adaptor enables GFP fluorescence to be examined using a typical dissecting microscope (eg Nikon)
StereoZoom Microscope 83604 Set with 10X Wide Field Eyepieces and Universal Boom Stand	Nikon	SMZ 745
Thermal Cycler	BioRad	T100
Trizol reagent	Life Technologies	15596018	CAUTION; Alternative RNA extraction methods can be used. http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf
Tween 20	Amresco	0777-1L