DNA Elektroporation, Isolering og Imaging af -myofibre
Summary
This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.
Abstract
Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.
Introduction
Individuelle myofibre er store, meget organiseret syncytial celler. Der er et par celle-kultur modeller for muskel; Men disse modeller har deres største begrænsning, at de ikke fuldt ud differentiere til modne myofibre. For eksempel er C2C12 og L6 cellelinjer afledt fra mus og rotter, henholdsvis 1-4. Under betingelser af serum sult, de mononukleære "myoblast-lignende" celler ophøre proliferation, undergår celle tilbagetrækning cyklus, og indgå den myogene program danner flerkernede celler med sarkomerer, kaldet myotuber. Med langvarige dyrkningsbetingelser kan myorør udvise kontraktile egenskaber og "spjæt" i kultur. Humane cellelinier har nu også etableret 5. Ud over disse immortaliserede cellelinjer kan mononukleære myoblaster isoleres fra muskel og under de samme betingelser af serum sult vil danne myotuber. Disse cellelinier og primære kulturer myoblast er meget brugerful fordi de kan transficeres med plasmider eller transduceret med vira og anvendes til at undersøge grundlæggende cellebiologiske processer. Men disse celler, selv når induceret til dannelse af myorør, mangler mange af de vigtigste træk ved moden muskel organisation. Specifikt myorør er meget mindre end de enkelte modne myofibre og mangler den normale form af myofibre. Kritisk, myorør mangler tværgående (T-) tubuli, den membranøs netværk kræves til effektiv Ca 2+ frigivelse hele myoplasm.
En alternativ metode til primær myoblast eller myogene cellelinjer indebærer anvendelse modne myofibre. Transgenese kan anvendes til at etablere ekspression af mærkede proteiner, men denne metode er kostbar og tidskrævende. In vivo elektroporation af musemuskel er dukket op som en foretrukken fremgangsmåde til dets hastighed og pålidelighed 6-10.
Fremgangsmåder til in vivo elektroporation og effektiv isolering af myofibre have blevet optimeret til musen flexor digitorum brevis (FDB) muskel 6. Fremgangsmåderne kan gennemføres let og er minimalt invasiv at inducere in vivo-ekspression fra plasmider. Denne fremgangsmåde er nu kombineret med høj opløsning billeddannelse metoder, herunder billeddannelse efter laser afbrydelse af sarcolemma 6,7. Kombinationen af fluorescerende farvestoffer og ekspression af fluorescensmærkede proteiner kan anvendes til at overvåge cellebiologiske processer i modne myofibre.
Protocol
Representative Results
Discussion
Anvendelsen af elektroporation til at studere fluorescens mærkede proteiner in vivo er velegnet til undersøgelse af muskel grund af manglen på passende cellelinje modeller, der trofast replikerer hele cellestruktur muskel. Denne protokol beskriver udnyttelsen af elektroporation og høj opløsning konfokal mikroskopi til at give detaljerede billeddannelse af protein lokalisering og translokation efter laser såret. Denne metode kan tilpasses til at studere andre cellulære processer, herunder cytoskelet…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Institutes of Health giver NS047726, NS072027 og AR052646.
Materials
| DMEM+A2:D32D42A2:D31AA2:D29 | Life Technologies | 11995-073 | Dissection Media Dilution: 50ml +100g BSA |
| Collagenase, Type II | Life Technologies | 17101-015 | Fiber Digestion Dilution: 160mg / 4ml DMEM (stock 40mg/ml aliquot) |
| Falcon 12-well dishes | Fisher Scientific | 877229 | Fiber Digestion |
| Ringers Solution | Fiber Digestion and Imaging Dilution: 146 mM NaCl 5 mM KCl 2 mM CaCl2 1 mM MCl2 10mM HEPES pH7.4 in H2O |
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| 1ml Pipettes | Axygen | T-1000-C-R | Digestion |
| Razor Blades | Personna | 94-120-71 | Digestion |
| Precision Glide 30G needle | BD Biosciences | 305128 | Dissection |
| 1x PBS (-/-) | Life Technologies | 14190-250 | Dissection |
| Sylgard 184 | Fisher Scientific | 50-366-794 | Dissection |
| Forceps | FST by Dumont | #5/45 | Dissection |
| Forceps | Roboz | RS-4913 | Dissection |
| Scissors | World Precision Instruments | 500260 | Dissection |
| BSA | Sigma | A7906 | Dissection media Dilution: 100mg / 50ml DMEM |
| Falcon 60mm dishes | Fisher Scientific | 08772B | Dissection- used to hold sylgard |
| Clay | Electroporation | ||
| Stimulator | Grass | s88x | Electroporation |
| Clamps | Radio Shack | 270-356 | Electroporation |
| Triadine | Aplicare | 82-220 | Electroporation |
| Precision Glide 27G needle | BD Biosciences | 305136 | Electroporation |
| Simulator | Grass | SIU-V | Electroporation |
| Gauze | Covidien | 2146 | Electroporation |
| Hyaluronidase | Sigma | H4272 | Injection Dilution: Add 160ul PBS (1X) to 40µl 5x stock |
| 1cc U-100 Insulin Syringe (28G1/2) | BD Biosciences | 329420 | Injection |
| Eppendorf Tubes | Fisher Scientific | 02-682-550 | Injection |
| 35mm glass bottom Microwell dish | MaTek | P35G-1.5-14-C | Microscopy |
| FM 4-64 | Life Technologies | T-13320 | Microscopy Dilution: Final 2.5 ug/ml |
| FM 1-43 | Life Technologies | T-35356 | Microscopy Dilution: Final 2.5 ug/ml |
| Endotoxinfree Plasmid Maxi Kit | Qiagen | 12362 | Plasmid Purification |
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