Name: the c. elegans intestine as a model for intercellular lumen morphogenesis and in vivo polarized membrane biogenesis at the single-cell level: labeling by antibody staining, rnai loss-of-function analysis and imaging
Uploaded: 2017-10-03T13:00:00
Description: Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

हम मारियो de नि । (MRC आणविक जीवविज्ञान, कैंब्रिज, ब्रिटेन की प्रयोगशाला), केनेथ जे Kemphues (कॉर्नेल विश्वविद्यालय, इथाका, संयुक्त राज्य अमरीका), मिशेल Labouesse (Institut de Biologie पेरिस महाजाल, विश्विद्यालय पियरे एट मैरी क्यूरी, पेरिस, फ्रांस) धंयवाद, Grégoire Michaux (विश्विद्यालय डेरेन 1, रेन, फ्रांस) और CGC, रिसर्च इन्फ्रास्ट्रक्चर प्रोग्राम (NIH P40) के OD010440 कार्यालय द्वारा वित्तपोषित, उपभेदों और एंटीबॉडी के लिए । यह काम पलाश NIH GM078653 द्वारा समर्थित था, MGH २२४५७० है और साे २२३८०९ को V.G.

1. लेबलिंग C. एलिगेंस आंत नोट: निकालनेवाला नहर के विश्लेषण पर लेखकों द्वारा संलग्न कागज देखें tubulogenesis < सुप वर्ग = "xref" > 2 ऊतक विशिष्ट फ्लोरोसेंट मार्कर के निर्माण के लिए plasmids और ट्रांसजेनिक ?...

<ol>
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Protoc. 6, 498-520 (2013).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+and+design+of+genetic+screens:+caenorhabditis+elegans.">The art and design of genetic screens: caenorhabditis elegans.</a> Nat. Rev. Genet. 3, 356-369 (2002).</li><li>Lee, Y. U., Son, M., Kim, J., Shim, Y. H., Kawasaki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CDC-25.2,+a+C.+elegans+ortholog+of+cdc25,+is+essential+for+the+progression+of+intestinal+divisions.">CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.</a> Cell Cycle. 15 (5), 654-666 (2016).</li><li>Hall, D. 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इस प्रोटोकॉल का वर्णन कैसे मानक हानि का गठबंधन करने के लिए समारोह आनुवंशिक/RNAi और इमेजिंग (लेबलिंग और सूक्ष्म) को vivo में दृश्य और आणविक विच्छेदन के लिए एक मॉडल के रूप में सी. एलिगेंस आंत्र उपकला का लाभ ल?...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td>Antibody staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>poly-L-lysine</td>
			<td>Sigma</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr >
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A949SK-4</td>
			<td></td>
		</tr>
		<tr >
			<td>Tween</td>
			<td>Fisher Scientific</td>
			<td>50-213-612</td>
			<td></td>
		</tr>
		<tr >
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td>Powdered milk</td>
			<td>Sigma</td>
			<td>MT409-1BTL</td>
			<td></td>
		</tr>
		<tr >
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>MH27 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:20 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>MH33 (mouse)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:10 Resources: Developmental Studies Hybridoma Bank.</td>
		</tr>
		<tr >
			<td>anti-ICB4 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:5 Resources: A gift from MariodeBono (Medical Research Council, England)</td>
		</tr>
		<tr >
			<td>anti-PAR-3 (rabbit)</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:50 Resources: A gift from Kenneth J. Kemphues (Cornell University)</td>
		</tr>
		<tr >
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Alexa Floor 568 (anti-rabbit)</td>
			<td>ABCam</td>
			<td>AB175471</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>Cy5 (anti-mouse)</td>
			<td>Life technologies</td>
			<td>A10524</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>TRITC (anti-rabbit)</td>
			<td>Invitrogen</td>
			<td>T2769</td>
			<td>Concentration: 1:200</td>
		</tr>
		<tr >
			<td>FITC (anti-mouse)</td>
			<td>Sigma</td>
			<td>F9006</td>
			<td>Concentration: 1:100</td>
		</tr>
		<tr >
			<td>Labeled chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Texas Red-Phalloidin</td>
			<td></td>
			<td></td>
			<td>Concentration: 1:100 Resources: Molecular Probes-T7471</td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Vacuum Grease Silicone</td>
			<td>Beckman</td>
			<td>335148</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>4448</td>
			<td></td>
		</tr>
		<tr >
			<td>Microscope coverslips (22x22-1)</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans related</td>
			<td></td>
			<td></td>
			<td>see reference29 for standardC. elegans culture and maintenance procedures.</td>
		</tr>
		<tr >
			<td>LB Medium and plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0116</td>
			<td></td>
		</tr>
		<tr >
			<td>Tetracycline</td>
			<td>Fisher Scientific</td>
			<td>BP912</td>
			<td></td>
		</tr>
		<tr >
			<td>M9 Medium</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>NGM plates</td>
			<td></td>
			<td></td>
			<td>see reference29 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>RNAi plates</td>
			<td></td>
			<td></td>
			<td>see reference30 for protocols.</td>
		</tr>
		<tr >
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr >
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr >
			<td>Tryptone</td>
			<td>Acros Organics</td>
			<td>611845000</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214040</td>
			<td></td>
		</tr>
		<tr >
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr >
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr >
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr >
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr >
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr >
			<td>IPTG</td>
			<td>US Biological</td>
			<td>I8500</td>
			<td></td>
		</tr>
		<tr >
			<td>Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>BP2648</td>
			<td></td>
		</tr>
		<tr >
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>50371500</td>
			<td></td>
		</tr>
		<tr >
			<td>Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>OP50 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>HT115 bacteria</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Genome-wide RNAi libraries Ahringer genome-wide RNAi feeding library (ref 30,49,50)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>C. elegans ORF-RNAi feeding library (ref51)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Imaging related</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP9221-500</td>
			<td></td>
		</tr>
		<tr >
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ-U</td>
			<td></td>
		</tr>
		<tr >
			<td>dissecting microscope equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions)</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr >
			<td>Laser-scanning confocal microscope</td>
			<td>Leica Microsystem</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr >
			<td>laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the c. elegans intestine as a model for intercellular lumen morphogenesis and in vivo polarized membrane biogenesis at the single-cell level: labeling by antibody staining, rnai loss-of-function analysis and imaging

कोशिकीय ट्यूबों, सभी आंतरिक अंगों की बुनियादी इकाइयों, एक दूसरे और/या extracellular मैट्रिक्स से संपर्क लुमेन और basolateral झिल्ली अस्तर शिखर झिल्ली के साथ, ध्रुवीकरण उपकला या endothelial कोशिकाओं से बना रहे हैं. कैसे इस विशिष्ट झिल्ली विषमता स्थापित है और अंग morphogenesis के दौरान बनाए रखा है अभी भी सेल जीवविज्ञान का एक अनसुलझे सवाल है । इस प्रोटोकॉल का वर्णन करता है C. एलिगेंस आंत ट्यूब morphogenesis के दौरान ध्रुवीय झिल्ली के विश्लेषण के लिए एक मॉडल के रूप में, शिखर झिल्ली और लुमेन पर जोर के साथ उत्पत्ति । C. एलिगेंस बीस सेल एकल-स्तरित आंत्र उपकला एक सरल द्विपक्षीय सममित ट्यूब में व्यवस्थित है, एक एकल सेल स्तर पर विश्लेषण की अनुमति । झिल्ली ध्रुवीकरण जल्दी embryogenesis के दौरान ध्रुवीकरण सेल विभाजन और प्रवास के साथ concomitantly होता है, लेकिन de नोवो ध्रुवीकरण लार्वा विकास भर में जारी है, जब कोशिकाओं अब पैदा और कदम नहीं है । बाद सेटिंग एक अलग उपसेलुलर परिवर्तन है कि एक साथ इन अलग ध्रुवीकरण प्रक्रियाओं, मुश्किल सबसे ध्रुवीयता मॉडल में भेद करने के लिए मध्यस्थता करने के लिए अनुमति देता है । शिखर-, basolateral झिल्ली-, जंक्शन-, cytoskeletal-और endomembrane घटकों लेबल और GFP फ्यूजन प्रोटीन द्वारा विकास भर में ट्रैक किया जा सकता है, या सीटू एंटीबॉडी धुंधला में द्वारा मूल्यांकन । जीव आनुवंशिक बहुमुखी प्रतिभा के साथ साथ, C. एलिगेंस आंत इस प्रकार दृश्य, विकास के लिए vivo मॉडल में एक अनूठा प्रदान करता है, और ध्रुवीय झिल्ली और ट्यूब की उत्पत्ति के आणविक आनुवंशिक विश्लेषण. विशिष्ट विधियों (सभी मानक) यहां वर्णित कैसे करने के लिए: लेबल आंतों एंटीबॉडी धुंधला द्वारा सेलुलर अवयव; विश्लेषण के नुकसान-समारोह आम तौर पर आवश्यक tubulogenesis जीन के लिए अनुकूलित अध्ययन द्वारा ध्रुवीकरण झिल्ली में शामिल जीन श्लेष; विभिन्न विकासात्मक चरणों के दौरान ध्रुवीकरण दोषों का आकलन करें; epifluorescence द्वारा phenotypes व्याख्या, विभेदक हस्तक्षेप कंट्रास्ट (उद्योग) और फोकल माइक्रोस्कोपी; दृश्य दोष यों तो । इस प्रोटोकॉल अक्सर अत्यधिक संरक्षित उपकला ध्रुवीयता, झिल्ली में शामिल अणुओं में से किसी का विश्लेषण करने के लिए अनुकूलित किया जा सकता है उत्पत्ति, ट्यूब और लुमेन morphogenesis.

इस प्रोटोकॉल का वर्णन कैसे आणविक विश्लेषण और एक कोशिका और सेलुलर स्तर पर सी. एलिगेंस आंत में, ध्रुवीय झिल्ली की उत्पत्ति और लुमेन morphogenesis कल्पना । बीस सेल एकल स्तरित सी. एलिगेंस आंत के म?...

Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

पारदर्शी सी एलिगेंस आंत कोशिकीय tubulogenesis के दौरान एकल सेल और उपसेलुलर स्तर पर apicobasal झिल्ली और लुमेन के अध्ययन के लिए एक "vivo में ऊतक चैंबर" के रूप में सेवा कर सकते हैं । इस प्रोटोकॉल का वर्णन कैसे मानक लेबलिंग, नुकसान की-समारोह आनुवंशिक/RNAi और सूक्ष्म दृष्टिकोण गठबंधन करने के लिए एक आणविक स्तर पर इन प्रक्रियाओं टुकड़े ।

सी. एलिगेंस आंत एक मॉडल के रूप में एक सेलुलर लुमेन Morphogenesis के लिए और Vivo में एक-कोशिका स्तर पर ध्रुवीकरण झिल्ली की उत्पत्ति: लेबलिंग द्वारा एंटीबॉडी धुंधला, RNAi के नुकसान का-समारोह विश्लेषण और इमेजिंग

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

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The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

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सी. एलिगेंस निकालनेवाला नहर Intracellular लुमेन Morphogenesis के लिए एक मॉडल के रूप में और Vivo में एक एकल कक्ष में ध्रुवीकरण झिल्ली की उत्पत्ति: लेबलिंग द्वारा GFP-फ्यूजन, RNAi इंटरेक्शन स्क्रीन और इमेजिंग

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

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