स्तनधारी कंकाल का उपयोग स्नायु के डीएनए अभिकर्मक Vivo में Electroporation
Summary
हम रहते electroporation और प्रोटीन अभिव्यक्ति का उपयोग कर प्रतिदीप्ति माइक्रोस्कोपी के बाद दृश्य का उपयोग कर चूहों के पैर की मांसपेशियों के तंतुओं में प्लास्मिड डीएनए के कुशल अभिकर्मक के लिए विस्तृत प्रक्रिया का वर्णन हैं.
Abstract
A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.
Protocol
FDB और कब मांसपेशियों के vivo electroporation में के लिए प्रायोगिक प्रक्रिया Vivo electroporation प्रोटोकॉल में शुरू करने से पहले, स्तनधारी अभिव्यक्ति plasmids 2-5 μg प्लाज्मिड / ते के μl नोट की रेंज में एकाग्रता उपज प?…
Discussion
हम यहाँ विस्तृत कदम का वर्णन है कि क्रम में कंकाल की मांसपेशी फाइबर में द्वारा vivo electroporation में डीएनए plasmids के प्रभावी transfections प्राप्त पालन किया जाना चाहिए . हमारे दृष्टिकोण का मुख्य लाभ के कार्यान्वयन की…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम हमारे साथ TPLSM सुविधा साझा करने के लिए, तंत्रिका जीव विज्ञान, UCLA के विभाग, डॉ. टी. ओटिस धन्यवाद, डॉ. सी. Fahlke, फिजियोलॉजी संस्थान, RWTH आकिन, जर्मनी, pEYFP – ClC1 प्लाज्मिड की तरह दान के लिए, और श्री तकनीकी सहायता के लिए आर. Serrano. इस काम के NIH / NIAMS AR047664 और AR54816 अनुदान से अनुदान द्वारा समर्थित किया गया.
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Tags
DNA TransfectionMammalian Skeletal MusclesIn Vivo ElectroporationCell BiologyTransgenic ProteinsFluorescently Tagged ConstructsMutant VariantsIn Vivo TransfectionPlasmidsLiverSkeletal MuscleBrainFlexor Digitorum BrevisInterosseus MusclesAdult MiceExperimental ParametersSoluble ProteinsStructural ProteinsMembrane ProteinsEGFPECFP3CalpainFKBP12β2a-DHPRMinidystrophinα-actininα1s-DHPRRyR1Cardiac Na/Ca2+ ExchangerNaV1.4 Na ChannelSERCA1
Cite This Article
DiFranco, M., Quinonez, M., Capote, J., Vergara, J. DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation. J. Vis. Exp. (32), e1520, doi:10.3791/1520 (2009).