Name: isolation of drosophila melanogaster testes
Uploaded: 2011-05-13T13:00:00
Duration: 3 min 13 s
Description: Watch this Scientific Journal Video about Isolation of Drosophila melanogaster Testes at JoVE.com

The authors thank Vasia Vagin for introducing this technique into our laboratory.

<ol>
	<li>Make a glass microscope slide with a black background: Cut 20# white paper to the same size as the microscope slide. Color one side of the slide-size paper black using a permanent marker (e.g., Sharpie). Apply white glue (Elmer's) to the black side and adhere the paper to the slide. The slide can be reused. Using &frac34;" label tape (Fisherbrand), secure the microscope slide, glass side up, to the CO2 fly pad to fix it in place.</li>
	<li>Collect 0-3 day old male flies.</li>
	<li>Prepare a bucket of ice, phosphate-buffered saline (PBS), dissecting needles.</li>
	<li>Anaesthetize 50-100 flies using a stream of CO2and transfer them from the bottle or vial to the fly pad near the microscope slide.</li>
	<li>Pipette ~50 &mu;l ice-cold PBS in a drop in the middle of the microscope slide.</li>
	<li>Gently pick up a male fly with the dissecting needle&mdash;dip the dissecting needle into PBS, gentle touch the ventral side (i.e., near the wings) of the fly with the needle&mdash;place it into the drop of PBS.</li>
	<li>Place one needle on the middle of the fly, between the thorax and the abdomen, pushing down firmly. Place the other needle between the sixth (A6) and seventh (A7) abdominal segments (the second and third stripes from the bottom); pull the abdomen away from the thorax.</li>
	<li>Tease the testes apart from other organs, especially the gut. Especially in white mutants, testes and gut look similar. Key differences are that testes always come in pairs; each testis has a spiral pattern and is more white in color than gut.</li>
	<li>Store the testes in ice-cold PBS in a polypropylene microcentrifuge tube.</li>
</ol>
Representative Results: 
<img src="/files/ftp_upload/2641/2641fig1.jpg" alt="Figure 1" /> 
Figure 1. Dorsal view of the male reproductive system,from flybase.org/reports/FBim0000074.html. Flybase attribution: "After Figure 38 A on page 508 from Miller (1950). A. Miller. The internal anatomy and histology of the imago of Drosophila melanogaster. In M. Demerec, editor, Biology of Drosophila, chapter 6, pages 420-534. Cold Spring Harbor Laboratory Press, facsimile edition, 1994. Copyright 1994 by Cold Spring Harbor Laboratory Press. Used with kind permission from Cold Spring Harbor Laboratory (CSHL) Press. See <a href="http://www.cshlpress.com">http://www.cshlpress.com</a>."

<ol>
	<li>Singh, S. R., Hou, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistological+Techniques+for+Studying+the+Drosophila+Male+Germline+Stem+Cell.">Immunohistological Techniques for Studying the Drosophila Male Germline Stem Cell.</a> Methods Mol Biol. 450, 45-59 (2008).</li></ol>

No conflicts of interest declared.

We provide a protocol to isolate testes from adult maleDrosophila melanogaster. Because the technique requires coordinating one's two hands, skill comes only with practice.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Dissecting needle</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>S17346</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slides, Plain </td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>12-553</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Light</td>
<td>Tool</td>
<td>Schott-Fostec</td>
<td>KO-1500</td>
<td>&nbsp;</td>
<tr>
<td>CO2 pad</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-172</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Flowbuddy Complete System</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-122WCU</td>
<td>&nbsp;</td>
<tr>
<td>Bubble Humidifier</td>
<td>Tool</td>
<td>Salter Labs</td>
<td>7100</td>
<td>&nbsp;</td>
<tr>
<td>Ball filler needle</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>54-119</td>
<td>&nbsp;</td>
<tr>
<td>&frac34;&#x201D; Label Tape</td>
<td>Supply</td>
<td>Fisher Scientific</td>
<td>15-901-5B</td>
<td>&nbsp;</td>		</tbody>
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isolation of drosophila melanogaster testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Watch this Scientific Journal Video about Isolation of Drosophila melanogaster Testes at JoVE.com

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

isolation-of-drosophila-melanogaster-testes

Research

JoVE Journal

Biology

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In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

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Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

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Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

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Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

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Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...