作者谨感谢布卢明顿股票中心，利群罗，查尔斯·朱克和莉莉和裕侬扬慷慨分享飞股票及以下资金：肉体-URM（以JD和SW），本科福特学院暑期奖学金（申银万国），BD公司暑期研究奖学金（以CL和DL），该蕾妮和Anthony M.马龙，MD &#39;63暑期研究奖学金（以DL），詹姆斯·&#39;75和简Colihan暑期研究奖学金（TO）通过校友/圣十字学院的家长暑期研究基金和Stransky基金会暑期研究奖学金（为TO）。特别感谢生物系和教务处在学院的圣十字对实验室全部工作的支持。

1.十字到生成MARCM苍蝇<ol><li>保持苍蝇玉米面糖蜜培养基（6.5克/升琼脂; 23.5克/升酵母; 60 g / L的玉米面; 60毫升/升糖蜜; 4毫升/升酸混合物; 0.13％Tegosept）或标准果蝇介质在22摄氏度在12小时的光照/黑暗循环。 注：比标准温度下是用来保持MARCM-股健康。这延伸到14天蛋和成年之间的发育时间。 </li><li>从一个MARCM就绪库存 - （8日龄约1）含有必要的遗传元件（GFP的上游活化序列（UAS-GFP）的GAL80阻遏蛋白，该Gal4的转录因子，的控制下使用处女雌性热休克驱动的重组，和FRT重组位点）。 注：我们MARCM现成的个股包括以下基因型：elavGal4，UAS-CD8-GFP，HS-flipase; FRT40A，tubGal80 / CYO; tubGal4 / TM3Sb。 注意：特定FRT位点，将取决于突变的位置在基因组中。例如，FRT40A用于左臂2号染色体，如NOMPC（见10的更多详细信息MARCM协议）测试突变。 </li><li>交叉MARCM就绪处女雌性到男性包含感兴趣的突变和其相应的FRT位点（例如，W-; NOMPC3，FRT40A / CYO + / +）。为了控制有效MARCM感应，设置使用雄性苍蝇含有相同FRT位点的第二交叉，但没有突变。使用5位女性的大致比例：1男在十字架。 </li></ol> 2.定时产卵<ol><li>使苍蝇交配用于至少一个晚上后，移动大人到新的小瓶中，用新鲜培养基。允许苍蝇之前再次转移杂交到新的小瓶产卵在黑暗环境中进行大约为4小时。注意产蛋期的开始和结束时间。 注意：当似乎有高数量的媒体卵经4小时interva结束之前更短的时间间隔是可以接受的湖<ol><li>保持小瓶鸡蛋足够数量（大约大于20），并存储在一个12小时的光/暗周期在22摄氏度至晚热冲击。 注意：小瓶用鸡蛋的介质上一个数低（小于大约20）是不可能产生足够数量的成蝇对于行为测试和效率起见，建议他们不被保留用于进一步的测试。 注：我们的苍蝇就提出用灯光从早上8点到晚上8点。我们已经找到了傍晚的时候分的领先优势，以更高的蛋密度，因此通常执行4之间定时十字 - 晚上8点到晚上8点 - 午夜。苍蝇奠定在室温下的鸡蛋放在一个黑暗的橱柜，然后返回到孵化第二天，以免干扰常规明暗周期在孵化器。 </li></ol></li></ol> 3.执行热休克来诱导有丝分裂重组<ol><li>大约85 - 产蛋后100小时，地点vIALS进入水浴在37摄氏度下1小时。确保水位到达媒体在小瓶的高度以上，但不完全淹没小瓶以防止幼虫溺水。 </li><li>从水浴中取出药瓶，并允许在室温下1小时的恢复期。 </li><li>地方小瓶放回37摄氏度的水浴中1小时。 </li><li>取下水浴和存储小瓶在孵化器12小时光照/黑暗周期在22摄氏度左右，直至羽化。 </li></ol> 4.准备行为测试<ol><li>羽化之后，麻醉苍蝇在冰上。选择苍蝇与包含所有为MARCM遗传元件和表示GFP表达标记的克隆很可能会根据在父个股中使用的可观察到的表型的标记物观察到的基因型。斩首苍蝇与虹膜切除术剪刀。 注：例如，我们选择了苍蝇不遗传标记CYO（大翅膀）和Sb（茬毛）来鉴定FY苍蝇，将包含所有必要创造马赛克区域的遗传元件标有绿色荧光蛋白。 注：斩首是既要防止成年人，当刺激飞走。 </li><li>放置无头苍蝇在封闭，潮湿的环境，并允许约10 - 20小时的恢复。只有用苍蝇这种权利本身扰动时进行进一步的测试。 30小时断头内的测试飞行。 </li></ol> 5.行为测试<ol><li>通过荧光解剖显微镜下观察断头苍蝇，发现标有绿色荧光蛋白的猪鬃外部感觉器官纯合子克隆。记录刷毛名称和左，右两侧。以消除任何潜在的偏见，该实验室构件进行刷毛刺激应盲目刷毛，野生型或突变体的基因型。 </li><li>通过偏转GFP引发疏导反射标塞得满满的向飞机身采用了硬毛或细镊子，观察腿再sponse。给苍蝇解除他们的腿在应对猪鬃刺激和苍蝇不动自己的腿得分为零分之一。 注：以前的研究6,7有特点的是通过刺激特定的刷毛引起腿部的反应。这包括，响应各刷毛的腿和苍蝇该特定刷毛刺激响应的比例。我们只打进了我们预期的基础上Vandervorst的分类6响应腿的动作。 </li><li>开展间隔2分钟间隔五位审判。 </li></ol>

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作者有没有竞争经济利益。

这个协议利用一个成人行为分析以筛选影响机械感觉在果蝇的突变。因为突变体的集合包含致死对元件的突变，将排除筛选作为成年人，该协议使得使用第一Lee和罗述的复杂的遗传技术的，（1999年）和详细的协议吴，罗，（2006年），以规避成人杀伤力。 MARCM诱导同源染色体之间有丝分裂重组以产生纯合细胞的克隆的区域。因为阻遏蛋白，浴盆-GAL80中有丝分裂重组发生的损失的这些?...

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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	<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>
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			<td height="21" style="height:21px;width:163px;">Water bath</td>
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			<td height="21" style="height:21px;width:163px;">Ice bucket</td>
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	</tbody>
</table>

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

因为结构和功能同源性的哺乳动物内耳的毛细胞，即支配果蝇外部感觉器官的神经元提供机械感觉的研究的优良模型系统。这个协议描述在果蝇中，可用于识别与机械感觉干扰突变简单的触摸行为。一个macrochaete的触觉刺激刷毛上的苍蝇的胸部引出一清洁反射从第一或第三站。与机械传导（如NOMPC）干扰的突变，或者与反射弧的其他方面，可以抑制疏导响应。成人行为的传统画面就会错过这一发展过程中发挥重要作用的突变体。相反，该​​协议结合镶嵌分析与阻遏细胞标记物（MARCM）触摸屏，以允许纯合突变细胞中，只有有限的区域由EXPR生成并标分裂国家绿色荧光蛋白（GFP）的。通过测试MARCM克隆为异常的行为反应，有可能筛选的致死对元件的突变的集合以寻找涉及机械感觉新基因，将已经错过由更传统的方法。

此协议的成功在很大程度上依赖于可检验的苍蝇的总产率，热休克的有效性以诱导有丝分裂重组，并获得在果蝇以2分钟的间隔刺激一个健壮的行为反应的能力。鉴于胚胎的估计12.5％包含必要MARCM元素和成年人E关闭对GFP表达的潜在的估计的16.67％，这是关键的期间定时规定以获得高数鸡蛋。一个成功的定时的一个例子笼罩，将在该协议的其余部分被用来显示在?...

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

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</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 次观看.  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.