Name: laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc
Uploaded: 2010-04-30T17:00:00
Description: Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

The authors thank prof. Chiara Campanella and AMRA Center of Competence, University of Naples Federico II, Naples, Italy, for providing them with the use of the laser microdissector, and Mr Vincenzo Vicidomini for generous help in the 3D animation.

Part 1. Preparation of Drosophila Imaginal Wing Discs Subjected to Specific and Localized RNAi.
As biological material, we used Drosophila imaginal wing discs obtained from transgenic larvae subjected to the localized and specific gene silencing by means of the GAL4/UAS system 1. This larval progeny orginates from a genetic cross involving two parental lines: one carrying the GAL4 driver, the second the UAS responder. In addition to express GAL4 in a specific tempor...

<ol>
	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

With respect to their basal expression levels achieved in the anterior/posterior compartments of unsilenced wing discs, the activity of the selected gene X was found reduced to 40% upon silencing. In contrast, one of its putative targets (genes Y) was found significantly up-regulated (7 fold-increase), leading us to validate the hypothesis that it was negatively regulated by gene X.
We conclude that the above described experimental approach can successfully be applied to the validation of put...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DEPC water</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>W4502</td>
<td>&nbsp;</td>
<tr>
<td>RNase Zap</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R2020</td>
<td>&nbsp;</td>
<tr>
<td>TRI Reagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9424</td>
<td>&nbsp;</td>
<tr>
<td>SuperScript III</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18080093</td>
<td>&nbsp;</td>
<tr>
<td>Master Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11761100</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl alcohol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I9516</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7559</td>
<td>&nbsp;</td>
<tr>
<td>Dream taq</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>EP0701</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Fly strains
Drosophila UAS-silencing lines can be obtained from VDRC RNAi collection 2. A collection of GAL4-driver lines is available at the Bloomington Drosophila Stock Center (Indiana, USA).
Equipment
Required equipment includes Laser microdissector (Leica LMD6000), Coulter Microfuge 22R (Beckman), PCR (BIO-RAD My Cycler), Real Time PCR (BIO-RAD iQ5). 
Tools
Required tools include: glass wells, brush, microdissection forceps, hanging drop slides, insect pins mounted on syringe needles, metal frames with PET membrane, plastic tubes (50 ml, 05 ml, 0.25 ml).		</div>

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Research

JoVE Journal

Biology

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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

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

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...