Name: analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells
Uploaded: 2011-05-31T12:00:00
Description: Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

इस काम एच. Fölsch स्वास्थ्य (GM070736) के राष्ट्रीय संस्थानों से एक अनुदान द्वारा वित्त पोषित किया गया था. एस एफ आंग एक एक * स्टार ग्रेजुएट छात्रवृत्ति पुरस्कार द्वारा समर्थित किया गया था, और आरएस कांग रोग प्रशिक्षण कार्यक्रम के सेलुलर और आणविक आधार (GM8061) द्वारा समर्थित किया गया था

1. प्लाज्मिड डीएनए के अलगाव <ol><li> एक सिग्मा Aldrich endotoxin मुक्त maxiprep किट का प्रयोग निर्माता प्रोटोकॉल के अनुसार endotoxin मुक्त डीएनए तैयार है. इस किट हमारे लिए काम करता है, क्योंकि यह मज़बूती से डीएनए की तैयारी से किसी ...

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एक सफल microinjection प्रयोग के लिए सबसे महत्वपूर्ण कदम है और डीएनए की गुणवत्ता और शुद्धता और अपनी कोशिकाओं के polarity हैं. Polarized कोशिकाओं के बिना, अपने इंजेक्शन नियंत्रण पहले से ही VSVG mistargeted होगा और प्रयोग किया नहीं किया...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> गर्म मंच के साथ 200 माइक्रोस्कोप Axiovert </td><td> कार्ल Zeiss इंक </td><td> कस्टम आदेश </td></tr><tr><td> Injectman NI2 Femtojet micromanipulator </td><td> Eppendorf </td><td> कस्टम आदेश </td></tr><tr><td> Femtotips द्वितीय (Microinjection सुई) </td><td> Eppendorf </td><td> 930000043 </td></tr><tr><td> Microloader युक्तियाँ </td><td> Eppendorf </td><td> 930001007 </td></tr><tr><td> साफ 12 मिमी transwell फिल्टर का समर्थन करता है </td><td> Corning costar </td><td> 3460 </td></tr><tr><td> Endotoxin मुक्त प्लाज्मिड maxiprep किट </td><td> सिग्मा Aldrich </td><td> NA0400 </td></tr></tbody></table>

analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

इस अनुच्छेद के विवरण और polarized microinjection तकनीक का उपयोग करके उपकला कोशिकाओं में छोटे GTPases की overexpression विश्लेषण में शामिल प्रक्रियाओं.

प्लास्मिड के Microinjection द्वारा Polarized उपकला कोशिकाओं में छोटे GTPases के फंक्शन का विश्लेषण

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

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