Os autores gostariam agradecer o centro de Bloomington estoque, Liqun Luo, Charles Zuker, e Lily e Yuh Nung Jan para a partilha generosa de ações da mosca ea seguinte para financiamento: SOMAS-URM (de JD e SW), Bacharel Ford Faculdade de Verão Fellowship (a sudoeste), BD Corporação Verão Research Fellowship (a CL e DL), The Renee e Anthony M. Marlon, MD &#39;63 Verão Research Fellowship (a DL) James C. &#39;75 e Jane Colihan Verão Research Fellowship (TO) através os alunos / pais Fundo do College of the Holy Cross Research Verão e da Fundação Stransky Research Fellowship Verão (a TO). Um agradecimento especial ao Departamento de Biologia e Gabinete do Reitor da College of the Holy Cross para o apoio de todo o trabalho no laboratório.

1. Cruzes para gerar Flies MarcM <ol><li> Manter moscas em meios de fubá melaço (6,5 g / L de agar; 23,5 g / L de levedura; 60 g / L da farinha de milho; 60 ml / L de melaço; 4 ml / mistura de ácido L; 0,13% Tegosept) ou mídia Drosophila padrão a 22 graus Celsius num ciclo luz / escuro de 12 h. Nota: A Baixa de temperatura padrão é usado para manter MarcM-stocks saudável. Isso amplia o tempo de desenvolvimento entre ovo e idade adulta para 14 dias. </li><li> Use fêmeas virgens (cerca de 1 - 8 dias de idade) a partir de um estoque MarcM-pronto contendo os elementos genéticos necessários (GFP sob o controlo de uma sequência a montante activadora (UAS-GFP), a proteína repressora Gal80, o factor de transcrição Gal4, uma de choque térmico recombinase com motor, e um local de recombinação FRT). Nota: Nossa MarcM prontos-existências conter os seguintes genótipos: elavGal4, UAS-CD8-GFP, hs-flipase; FRT40A, tubGal80 / CYO; tubGal4 / TM3Sb. Nota: O local DRF específico dependerá da localização da mutaçãono genoma. Por exemplo, FRT40A é usado para testar as mutações no braço esquerdo do cromossoma 2, tal como NOMPC (ver 10 para mais detalhes sobre os protocolos MarcM). </li><li> Fêmeas virgens Cruz MarcM pronto para homens que contêm uma mutação de interesse e seu correspondente local DRF (por exemplo, w-; NOMPC3, FRT40A / CYO; + / +). Para controlar a indução MarcM eficaz, definir uma segunda cruzada usando as moscas macho contendo o mesmo local DRF, mas sem uma mutação. Use uma proporção aproximada de 5 fêmeas: 1 macho em cruzamentos. </li></ol> 2. Temporário postura de ovos <ol><li> Depois de permitir que as moscas para acasalar para pelo menos uma noite, mover adultos em novos tubos de ensaio com meio fresco. Permitir moscas para colocar ovos em um ambiente escuro por cerca de 4 horas antes de voltar a transferir as cruzes em novos frascos. Observe os horários de início e de término do período de postura de ovos. Nota: Intervalos mais curtos são aceitáveis ​​quando parece haver um elevado número de ovos sobre a mídia antes do final do interva 4-hreu. <ol><li> Mantenha os frascos com um número suficiente de ovos (aproximadamente superior a 20) e armazenar em um ciclo claro / escuro de 12 horas a 22 graus Celsius para ser a choque térmico posterior. Nota: Os frascos com um baixo número de ovos na mídia (menos de cerca de 20) não são susceptíveis de produzir um número suficiente de moscas adultas para o teste comportamental e no interesse da eficiência, recomenda-se não ser retidos por mais testes. Nota: Os nossos moscas são levantadas com as luzes acesas 08:00-20:00. Nós descobrimos que o tempo do entardecer pontos levam a densidades mais elevadas de ovos, por isso costumam realizar cruzamentos programados entre 4-8 pm e 8:00-meia-noite. As moscas põem os seus ovos à temperatura ambiente numa câmara escura e, em seguida, são devolvidos para a incubadora no dia seguinte, de modo a não perturbar o ciclo de luz-escuro normal na incubadora. </li></ol></li></ol> 3. Execute choque térmico para induzir Mitotic Recombinação <ol><li> Cerca de 85-100 horas após a postura dos ovos, lugar vIals num banho de água a 37 graus Celsius durante 1 hora. Certifique-se que o nível da água atinge acima da altura dos meios de comunicação nos frascos, mas não submergir totalmente os frascos para evitar que as larvas do afogamento. </li><li> Remova frascos de banho de água e permitir um período de recuperação de 1 hora em temperatura ambiente. </li><li> Colocar os frascos de volta para 37 graus Celsius banho de água durante 1 h. </li><li> Remova frascos de banho de água e guarde na incubadora em 12 horas ciclo claro / escuro, a 22 graus Celsius até eclosão. </li></ol> 4. Preparação para o Teste Comportamental <ol><li> Seguindo eclosão, anestesiar moscas no gelo. Selecione moscas com genótipos que contêm todos os elementos genéticos para MarcM e indicam clones marcados de expressão GFP provavelmente será observado com base nos marcadores fenotípicos observáveis ​​usados ​​nos estoques pai. Decapitar moscas com iridectomy tesoura. Nota: Por exemplo, foram selecionados para as moscas sem os marcadores genéticos CYO (asas chaves) e Sb (cerda restolho) a identimoscas fy que contêm todos os elementos genéticos necessários para criar regiões de mosaico marcadas com GFP. Nota: decapitação é necessário para evitar que os adultos de voar para longe quando estimulados. </li><li> Coloque moscas sem cabeça em um ambiente fechado, úmido e permitir que cerca de 10-20 horas de recuperação. Só uso voa esse direito-se quando perturbado por mais testes. Teste voa dentro de 30 horas de decapitação. </li></ol> 5. Teste Comportamental <ol><li> Ao observar moscas decapitados sob um microscópio de fluorescência de dissecação, os clones homozigóticos identificar marcadas com GFP nas cerdas órgãos sensoriais externas. Grave o nome de cerdas e do lado esquerdo ou direito. Para eliminar qualquer viés potencial, o membro do laboratório de realizar a estimulação de cerdas devem ser cego para o genótipo da cerda, do tipo selvagem ou mutante. </li><li> Provocar um reflexo aliciamento, desviando o GFP marcado cerda para o corpo da mosca com um cabelo duro ou uma pinça fina e observar a re pernasponse. Dê uma nota de um a moscas que levantar a perna em resposta à estimulação de cerdas e uma pontuação de zero a moscas que não se movem as suas pernas. Nota: Anterior estudos 6,7 caracterizaram a resposta perna que é provocada por estimular uma cerda particular. Isto inclui a perna que responde a cada cerda e a percentagem de moscas que respondem à estimulação de cerdas que em particular. Nós só marcou para os movimentos da perna que o esperado para responder baseado na classificação de Vandervorst 6. </li><li> Realizar cinco ensaios espaçados 2 min de intervalo. </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. 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Os autores não têm interesses financeiros concorrentes.

Este protocolo utiliza um ensaio comportamental adulto para o rastreio de mutações que afectam mechanosensation em Drosophila. Uma vez que a colecção de mutantes contém letais mutações P-elemento, que evitarão a rastreio como adultos, este protocolo faz uso de uma técnica genética complexa primeiro descrito por Lee e Luo , (1999) e detalhada como um protocolo por Wu e Luo, (2006) para contornar adulto letalidade. MarcM induz recombinações mitóticas entre cromossomos homólogos para gerar regiões c...

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.

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			<td height="39" style="height:39px;width:163px;">Molasses (1Gallon)</td>
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			<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>
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			<td height="40" style="height:40px;width:163px;">6oz Square Bottom Bottle (PP)</td>
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			<td height="21" style="height:21px;width:163px;">Flugs - Plastic Fly Bottles</td>
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			<td height="21" style="height:21px;width:163px;">Rayon Balls, Large</td>
			<td style="width:163px;">Genesee</td>
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			<td style="width:163px;">Fly Cultures</td>
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			<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>
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			<td height="21" style="height:21px;width:163px;">Droso-Filler, Bottles</td>
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			<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

Devido à homologia estrutural e funcional para as células ciliadas do ouvido interno de mamífero, os neurónios que enervam os órgãos dos sentidos de Drosophila externos proporcionam um excelente sistema modelo para o estudo de mechanosensation. Este protocolo descreve um comportamento simples toque na mosca da fruta, que podem ser utilizados para identificar as mutações que interferem com mechanosensation. A estimulação táctil de um macrochaete cerdas sobre o tórax de moscas provoca um reflexo da preparação a partir de qualquer da primeira ou terceira perna. As mutações que interferem com mecanotransdutores (tais como NOMPC), ou com outros aspectos do arco reflexo, pode inibir a resposta de preparação. Uma tela tradicional de comportamentos adultos teria perdido mutantes que têm papéis essenciais durante o desenvolvimento. Em vez disso, este protocolo combina a tela de toque com a análise do mosaico com um marcador de células repressible (MarcM) para permitir apenas regiões limitadas de células mutantes homozigotos para ser gerado e marcado pela exprESSÃO da proteína verde fluorescente (GFP). Ao testar clones MarcM para respostas de comportamento anormal, é possível rastrear um conjunto de mutações letais p-elemento de pesquisa para novos genes envolvidos no mechanosensation que teriam sido perdidas por métodos mais tradicionais.

O sucesso deste protocolo depende em grande medida o rendimento global de moscas testáveis, a eficácia do choque térmico para induzir a recombinação mitótica, e a capacidade de obter uma resposta comportamental robusto em moscas estimuladas em intervalos de 2 min. Dado que cerca de 12,5% de embriões conter os elementos necessários MarcM e estima-se que 16,67% da população adulta que EFeche têm o potencial para a expressão de GFP, que é fundamental para obter um elevado núme...

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

Clones em um ensaio comportamental para Mechanosensation de MarcM baseada em<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 ...

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Neuroscience

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

10.1K visualizações.  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.