Name: dna transfection of mammalian skeletal muscles using in vivo electroporation
Uploaded: 2009-10-19T14:00:00
Description: Watch this Scientific Journal Video about DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation at JoVE.com

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.

FDB और कब मांसपेशियों के vivo electroporation में के लिए प्रायोगिक प्रक्रिया <ol><li> 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?...

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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...

dna transfection of mammalian skeletal muscles using in vivo electroporation

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, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-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.

Watch this Scientific Journal Video about DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation at JoVE.com

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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.

स्तनधारी कंकाल का उपयोग स्नायु के डीएनए अभिकर्मक Vivo में Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

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Biology

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Dissociated वयस्क स्तनधारी न्यूरॉन्स में डीएनए की Intranuclear Microinjection

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

स्तनधारी कोशिकाओं में डीएनए डबल कतरा तोड़ मरम्मत (DSB) के विश्लेषण

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Morpholinos के vivo electroporation में वयस्क Zebrafish रेटिना में

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

पृथक कंकाल की मांसपेशियों में सिकुड़ना fatigability, और alternans के पूर्व vivo आकलन

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

कंकाल की मांसपेशियों के Isometric और सनकी सेना पीढ़ी आकलन पेशी अपविकास murine मॉडल से पृथक

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Ratiometric bimolecular संकेतकों का उपयोग जीवित कोशिकाओं में एकल इंजीनियर शाही सेना टेप के वास्तविक समय इमेजिंग

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In Vivo Microinjection and Electroporation of Mouse Testis

In Vivo Microinjection and Electroporation of Mouse Testis

माउस वृषण की विवो Microinjection और electroporation में

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

माउस कंकाल की मांसपेशी में जांच और डीएनए की क्षति के विश्लेषण<em&gt; बगल में</em&gt; TUNEL के विधि का प्रयोग

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

DNA Electroporation, Isolation and Imaging of Myofibers

डीएनए इलेक्ट्रोपोरेशन, Myofibers के अलगाव और इमेजिंग

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

स्तनधारी कोशिकाओं में नवजात डीएनए के लिए बाध्य प्रोटीन की परीक्षा BrdU चिप-खांचा-पश्चिमी तकनीक का प्रयोग

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...