Gli autori desiderano ringraziare il centro di Bloomington magazzino, Liqun Luo, Charles Zuker, e Lily e Yuh Nung Jan per la generosa condivisione delle scorte mosca e il seguente per il finanziamento: Somas-URM (JD e SW), Laurea Ford Facoltà Estate Fellowship (SW), BD Corporation Estate Research Fellowship (a CL e DL), Il Renee e Anthony M. Marlon, MD &#39;63 Estate Research Fellowship (DL) James C. &#39;75 e Jane Colihan Estate Research Fellowship (TO) attraverso Alumni / Parent Estate Fondo di ricerca del Collegio della Santa Croce e il Stransky Fondazione Estate Research Fellowship (a TO). Un ringraziamento speciale al Dipartimento di Biologia e l&#39;Ufficio di Presidenza a College of the Holy Cross per il sostegno di tutto il lavoro in laboratorio.

1. Croci per generare mosche marcm <ol><li> Mantenere mosche sulla melassa supporti farina di mais (6,5 g / l di agar; 23,5 g / L di lievito; 60 g / L di farina di mais, 60 ml / L melassa; 4 ml / miscela acido L; 0,13% Tegosept) o supporti Drosophila norma a 22 gradi Celsius in una luce 12 ore / ciclo scuro. Nota: A inferiore ad una temperatura standard viene utilizzato per mantenere marcm-scorte sano. Questo si estende il tempo di sviluppo fra l&#39;uovo e l&#39;età adulta a 14 giorni. </li><li> Utilizzare femmine vergini (circa 1 - 8 giorni di età) da uno stock marcm-ready contenente gli elementi genetici necessari (GFP sotto il controllo di una sequenza upstream-attivazione (UAS-GFP), la proteina repressore Gal80, il fattore di trascrizione Gal4, un shock termico ricombinasi guidato, e una ricombinazione sito FRT). Nota: Il nostro marcm ready-titoli contengono i seguenti genotipi: elavGal4, UAS-CD8-GFP, hs-flipase; FRT40A, tubGal80 / cyo; tubGal4 / TM3Sb. Nota: Il sito FRT specifico dipenderà dalla posizione della mutazionenel genoma. Ad esempio, FRT40A è utilizzato per testare le mutazioni sul braccio sinistro del cromosoma 2, come NOMPC (vedi 10 per maggiori dettagli sui protocolli marcm). </li><li> Croce marcm-ready femmine vergini per i maschi che contengono una mutazione di interesse e il suo sito FRT corrispondente (ad esempio, W-, NOMPC3, FRT40A / cyo; + / +). Per controllare per l&#39;induzione marcm efficace, impostare una seconda croce con le mosche di sesso maschile che contengono lo stesso sito FRT, ma senza una mutazione. Utilizzare un rapporto approssimativo di 5 femmine: 1 maschio in croci. </li></ol> 2. Timed ovaiole <ol><li> Dopo aver lasciato le mosche di accoppiarsi per almeno una notte, spostare gli adulti in nuove flaconcini con mezzi freschi. Lasciare le mosche di deporre le uova in un ambiente scuro per circa 4 ore prima di nuovo trasferire le croci in nuove fiale. Nota i tempi di inizio e di fine del periodo di deposizione delle uova. Nota: gli intervalli più brevi sono accettabili quando sembra che vi sia un alto numero di uova sui media prima della fine del interva di 4 orel. <ol><li> Tenere i flaconcini con un numero sufficiente di uova (circa maggiore di 20) e conservare in una luce 12 ore / buio ciclo a 22 gradi centigradi per essere il calore scioccato più tardi. Nota: Fiale con un basso numero di uova sul supporto (inferiore a circa 20) sono difficilmente danno un numero sufficiente di mosche adulte per i test comportamentali e nell&#39;interesse di efficienza si consiglia di non che essere conservati per ulteriori test. Nota: I nostri mosche sono allevati con le luci accese 08:00-20:00. Abbiamo trovato il tempo serata punti portano a densità più elevate di uova, quindi di solito eseguire incroci cronometrati tra 4-8 PM e 8:00 a mezzanotte. Le mosche depongono le loro uova a RT in un armadio buio ed sono restituiti al incubatore il giorno seguente in modo da non disturbare il normale ciclo luce-buio nell&#39;incubatrice. </li></ol></li></ol> 3. Eseguire Shock termico per indurre Mitotic Ricombinazione <ol><li> Circa 85-100 ore dopo la deposizione delle uova, luogo vIALS in un bagno d&#39;acqua a 37 ° C per 1 ora. Assicurarsi che il livello dell&#39;acqua raggiunge sopra l&#39;altezza media in fiale, ma non annulla completamente le fiale per evitare che le larve dall&#39;annegamento. </li><li> Rimuovere le fiale da bagno di acqua e consentire un periodo di recupero 1 ora a temperatura ambiente. </li><li> Luogo fiale indietro in 37 ° C bagnomaria per 1 ora. </li><li> Rimuovere le fiale da bagnomaria e conservare in incubatrice in luce 12 ore / buio ciclo a 22 ° C fino eclosion. </li></ol> 4. Preparazione per il test comportamentale <ol><li> A seguito di eclosion, anestetizzare mosche sul ghiaccio. Selezionare mosche con genotipi che contengono tutti gli elementi genetici per marcm e indicano probabilmente essere osservati GFP cloni di espressione-segnato in base ai marcatori fenotipici osservabili utilizzati nelle scorte genitori. Decapitare mosche con iridectomia forbici. Nota: Per esempio, abbiamo selezionato per le mosche, senza i marcatori genetici cyo (ali graffe) e Sb (setola stoppie) per identimosche fy che contengono tutti gli elementi genetici necessari per creare le regioni mosaico contrassegnati con GFP. Nota: La decapitazione è necessaria per evitare che le adulti di volare via quando stimolato. </li><li> Posizionare mosche senza testa in un ambiente umido chiuso e consentire circa 10 - 20 ore di recupero. Solo uso vola tale diritto se stessi quando perturbato per ulteriori test. Prova vola entro 30 ore della decapitazione. </li></ol> 5. Test comportamentale <ol><li> Osservando mosche decapitati sotto un microscopio a fluorescenza dissezione, identificare i cloni omozigoti contrassegnati con GFP ai setola organi sensoriali esterni. Registrare il nome setole e lato sinistro o destro. Per eliminare ogni pregiudizio potenziale, il membro del laboratorio di eseguire la stimolazione setole dovrebbe essere ciechi per il genotipo del setola, wild-type o mutante. </li><li> Suscitare una reflex grooming deviando la GFP contrassegnato setole verso il corpo volo con un capello rigido o una pinza sottile e osservare la ri gambarisposta. Dare un punteggio da uno a mosche che sollevare la gamba in risposta alla stimolazione setole e un punteggio pari a zero per le mosche che non si muovono le gambe. Nota: Precedente studi 6,7 hanno caratterizzato la risposta della gamba che si manifesta stimolando una particolare setola. Questo include la gamba che risponde ad ogni setola e la proporzione di mosche che rispondono alla stimolazione di quel particolare setola. Abbiamo segnato solo per i movimenti della gamba che ci aspettavamo di rispondere basò sulla classificazione di Vandervorst 6. </li><li> Condurre cinque studi distanziati 2 minuti a parte. </li></ol>

<ol>
	<li>Gr&#252;nert, U., Gnatzy, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K++and+Ca+++in+the+receptor+lymph+of+arthropod+cuticular+mechanoreceptors.">K+ and Ca++ in the receptor lymph of arthropod cuticular mechanoreceptors.</a> J Comp Physiol A. 161, 329-333 (1987).</li><li>Kernan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+and+auditory+transduction+in+Drosophila.">Mechanotransduction and auditory transduction in Drosophila.</a> Pflugers Arch. 454, 703-720 (2007).</li><li>Gillespie, P. G., Walker, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+mechanosensory+transduction.">Molecular basis of mechanosensory transduction.</a> Nature. 413, 194-202 (2001).</li><li>Corfas, G., Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+fatigue+of+a+mechanosensory+neuron+in+wild-type+Drosophila+and+in+memory+mutants.">Adaptation and fatigue of a mechanosensory neuron in wild-type Drosophila and in memory mutants.</a> J Neurosci. 10, 491-499 (1990).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12, 1195-1206 (1994).</li><li>Vandervorst, P., Ghysen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+sensory+connections+in+Drosophila.">Genetic control of sensory connections in Drosophila.</a> Nature. 286, 65-67 (1980).</li><li>Corfas, G., Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+and+dishabituation+of+a+cleaning+reflex+in+normal+and+mutant+Drosophila.">Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila.</a> J Neurosci. 9, 56-62 (1989).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3, e59 (2005).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+in+Drosophila.">A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila.</a> Nature Protocols. 1, 2583-2589 (2006).</li><li>Eberl, D. F., Hardy, R. W., Kernan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+similar+transduction+mechanisms+for+touch+and+hearing+in+Drosophila.">Genetically similar transduction mechanisms for touch and hearing in Drosophila.</a> J Neurosci. 20, 5981-5988 (2000).</li><li>Walker, R. G., Willingham, A. T., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mechanosensory+transduction+channel.">A Drosophila mechanosensory transduction channel.</a> Science. 287, 2229-2234 (2000).</li><li>Yan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+NOMPC+is+a+mechanotransduction+channel+subunit+for+gentle-touch+sensation.">Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation.</a> Nature. 493, 221-225 (2013).</li><li>Arnadottir, J., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+Mechanosensitive+Channels.">Eukaryotic Mechanosensitive Channels.</a> Annual Review of Biophysics. 39, 111-137 (2010).</li><li>Christensen, A. P., Corey, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP+channels+in+mechanosensation:+direct+or+indirect+activation?.">TRP channels in mechanosensation: direct or indirect activation?.</a> Nature Reviews Neuroscience. 8, 510-521 (2007).</li><li>Cheng, L. E., Song, W., Looger, L. L., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+TRP+Channel+NompC+in+Drosophila+Larval+and+Adult+Locomotion.">The Role of the TRP Channel NompC in Drosophila Larval and Adult Locomotion.</a> Neuron. 67, 373-380 (2010).</li><li>Lee, J., Moon, S., Cha, Y., Chung, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRPN(=NOMPC)+channel+localizes+to+the+distal+end+of+mechanosensory+cilia.">Drosophila TRPN(=NOMPC) channel localizes to the distal end of mechanosensory cilia.</a> PLoS One. 5, e11012 (2010).</li><li>Liang, X., Madrid, J., Saleh, H. S., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOMPC,+a+Member+of+the+TRP+Channel+Family,+Localizes+to+the+Tubular+Body+and+Distal+Cilium+of+Drosophila+Campaniform+and+Chordotonal+Receptor+Cells.">NOMPC, a Member of the TRP Channel Family, Localizes to the Tubular Body and Distal Cilium of Drosophila Campaniform and Chordotonal Receptor Cells.</a> Cytoskeleton. 68, 1-7 (2011).</li><li>de Vries, S. E., Clandinin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loom-sensitive+neurons+link+computation+to+action+in+the+Drosophila+visual+system.">Loom-sensitive neurons link computation to action in the Drosophila visual system.</a> Curr Biol. 22, 353-362 (2012).</li><li>Jarman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+mechanosensation+using+the+fly.">Studies of mechanosensation using the fly.</a> Hum Mol Genet. 11, 1215-1218 (2002).</li></ol>

Gli autori non hanno interessi finanziari concorrenti.

Questo protocollo utilizza un test comportamentale adulti per lo screening per le mutazioni che colpiscono mechanosensation in Drosophila. Poiché la collezione di mutanti contiene letali mutazioni p-elemento che escluderebbe di screening come gli adulti, questo protocollo fa uso di una tecnica genetica complessa descritta da Lee e Luo , (1999) e dettagliato come protocollo da Wu e Luo, (2006) per aggirare letalità degli adulti. Marcm induce ricombinazione mitotica fra cromosomi omologhi di generare regioni cl...

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Brewers Yeast (25 lb)</td>
			<td style="width:163px;">MP Biomedicals&#160;</td>
			<td style="width:163px;">ICN90331225&#160;</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Corn (25lb)</td>
			<td style="width:163px;">MP Biomedicals&#160;</td>
			<td style="width:163px;">ICN90141125&#160;</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Agar (1lb)</td>
			<td style="width:163px;">MoorAgar Inc.</td>
			<td style="width:163px;">41004</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Tegosept (5kg)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">20-259</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Molasses (1Gallon)</td>
			<td style="width:163px;">Sugarmill Brand - Thomsen Food Service</td>
			<td style="width:163px;">0 2625</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Propionic Acid</td>
			<td style="width:163px;">Fisher</td>
			<td style="width:163px;">A258-500</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Phosphoric acid</td>
			<td style="width:163px;">Fisher</td>
			<td style="width:163px;">A260-500</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Drosophila Vials, Narrow (PS)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">32-109</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">6oz Square Bottom Bottle (PP)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">32-130</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Flugs - Plastic Fly Bottles</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">49-100</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Rayon Balls, Large</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">51-100</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Droso-Filler, Narrow</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">59-168</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Droso-Filler, Bottles</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">59-170</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:163px;">8A-C / gear driven lab stirrer with c-clamp mount&#160; 1/15HP, 700RPM variable speed, 115V, 50/60Hz</td>
			<td style="width:163px;">Cleveland Mixer</td>
			<td style="width:163px;">8A-C</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Water jacketed Kettle</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Diurnal Growth Chamber</td>
			<td style="width:163px;">Forma Scientific</td>
			<td style="width:163px;"></td>
			<td style="width:163px;">Temperature and light/dark cycle controlled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Water bath</td>
			<td style="width:163px;">VWR</td>
			<td style="width:163px;"></td>
			<td style="width:163px;">For heat shock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">MicroScissors&#160;</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:163px;">&#160;15000-08</td>
			<td style="width:163px;">For removing heads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Fluroscence Dissecting Microscope</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">SteREO Discovery V8&#160;</td>
			<td style="width:163px;">With GFP cube (KSC295-814D) band pass filter</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Fluroscence Light Source</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">X-Cite 120</td>
			<td style="width:163px;">Fiber optic light pipe makes this easy to configure&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Camera for Scope</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">AxioCam ICc1</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Image acquistion software</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Ice bucket</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for cold anthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Homemade cold anthesia tray</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for cold anthesia decapitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Plastic boxes</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for recovery of decaptitated flies in humid environment</td>
		</tr>
	</tbody>
</table>

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

A causa della omologia strutturale e funzionale alle cellule ciliate dell&#39;orecchio interno dei mammiferi, i neuroni che innervano i Drosophila organi di senso esterni forniscono un eccellente sistema modello per lo studio della mechanosensation. Questo protocollo descrive un semplice tocco di comportamento nei moscerini della frutta che possono essere utilizzati per identificare le mutazioni che interferiscono con mechanosensation. La stimolazione tattile di una macrochaete setola sul torace di mosche provoca un riflesso governare sia dal primo o terza gamba. Le mutazioni che interferiscono con mechanotransduction (come NOMPC), o con altri aspetti della dell&#39;arco riflesso, possono inibire la risposta governare. Uno schermo tradizionale di comportamenti adulti avrebbe perso mutanti che hanno un ruolo essenziale durante lo sviluppo. Invece, questo protocollo combina il touch screen con analisi mosaico con un marker delle cellule reprimibile (marcm) per consentire solo le regioni limitate di cellule mutanti omozigoti per essere generato e segnata dalla exprESSIONE di proteina fluorescente verde (GFP). Testando cloni marcm per risposte comportamentali anomali, è possibile programmare una collezione di mutazioni p-elemento letali per la ricerca di nuovi geni coinvolti nella mechanosensation che sarebbero state perse con i metodi più tradizionali.

Il successo di questo protocollo dipende in gran parte la resa complessiva di mosche testabili, l&#39;efficacia dello shock termico per indurre ricombinazione mitotica, e la capacità di ottenere una risposta comportamentale robusta in linea stimolati ad intervalli di 2 min. Dal momento che si stima che il 12,5% degli embrioni contengono elementi marcm necessari e si stima che 16,67% degli adulti che Eclose hanno il potenziale per l&#39;espressione della GFP, è fondamentale per ottenere...

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Un test comportamentale per Mechanosensation di cloni in marcm-based<em&gt; Drosophila melanogaster</em

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

a-behavioral-assay-for-mechanosensation-marcm-based-clones-drosophila

Research

JoVE Journal

Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

10.1K Views.  College of the Holy Cross. In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.