Name: DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation (Video) | JoVE Journal
Uploaded: 2009-10-19T14:00:00.000+00:00
Duration: 15 min 56 s
Description: Learn in vivo electroporation for DNA transfection into mouse skeletal muscles using hyaluronidase and gold-plated needles. This method enables efficient gene delivery and fluorescence visualization for muscle biology research.

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5 minutes

We thank Dr. T. Otis, Department of Neurobiology, UCLA, for sharing the TPLSM facility with us, Dr. C. Fahlke, Institute of Physiology, RWTH Aachen, Germany, for the kind donation of the pEYFP-ClC1 plasmid, and Mr. R. Serrano for technical support. This work was supported by grants from NIH/NIAMS grants AR047664 and AR54816.

Experimental Procedures for in vivo electroporation of FDB and IO muscles
<ol>
<li>Before starting the in vivo electroporation protocols, mammalian expression plasmids must be amplified to yield concentration in the range of to 2-5 μg plasmid/μl of TE. Note: We routinely use commercial amplification kits and follow the manufacturer’ procedures. Commercial expression plasmids carrying the CMV promoter work very well in skeletal muscle in vivo transfections. </li>
<li>Aliquot the necessary volume (10-20 μl) of the plasmid solution and save it in two 0.5 ml Eppendorf tubes (one for each foot of the mouse).</li>
<li>Prepare a solution containing 2 mg/ml hyaluronidase in sterile Tyrode.</li>
<li>Using an anesthetizing box, deeply anesthetize a mouse using 4% isoflurane in O2 with an approved gas anesthetic machine. Place the animal on a heating pad (37 °C) and maintain the anesthesia using a rodent face mask. Monitor the anesthetic depth by toe pinch reflex.</li>
<li>Under observation with a dissection microscope, inject 10 μL of the hyaluronidase solution under the footpads of one foot of the mouse using a 1” long 33 gauge sterile needle. Penetrate the skin at a point close to the heel of the foot and advance the needle subcutaneously towards the base of the toes for ~1/4”.</li>
<li>Repeat the procedure with the other foot if so desired.</li>
<li>Disconnect the anesthesia and place the mouse in a cage. Allow it to fully recover from anesthesia.</li>
<li>After one hour, anesthetize the animal for a second time and place it on the heating pad. Following the same procedure described for the hyaluronidase solution, inject a total of 20-50 μg of the plasmid DNA (depending on the size of the plasmid construct). The total injection volume should be less than 20 μL/foot. Note: when 15-20 μL is necessary, it is advisable to close the skin at the needle entry point with tissue-glue.</li>
<li>Disconnect the anesthesia and place the mouse in a cage. Allow it to fully recover from anesthesia and wait for 10-15 min.</li>
<li>Anesthetize the animal for the third time and place it on the heating pad.</li>
<li>Select one foot of the animal. Place one gold-plated acupuncture needle under the skin at heel, and a second one at the base of the toes. Electrodes are oriented parallel to each other and perpendicular to the long axis of the foot.</li>
<li>Connect the head of the needles (electrodes) to the electrical stimulator using micro-clip connectors. Electroporate the muscles by applying 20 pulses, 20 ms in duration/each, at 1Hz. Depending on the spacing of the electrodes, the pulses’ voltage amplitude is adjusted (by monitoring with an oscilloscope) to yield an electric field of ~100 V/cm. Note: No contractions in response to the stimuli should be observed if the level of anesthesia is adequate. </li>
<li>If so desired, repeat the above procedures in the contralateral foot of the animal.</li>
<li>Return the animal to its cage and once fully recovered from anesthesia maintain it under observation. Note: if the procedure went normally, the animal should regain full mobility within 30 minutes and afterwards is ready to be sent back to the animal room at the vivarium. The injections of hyaluronidase and DNA in the footpads do not have noticeable adverse effects on the animals.
Once recovered from anesthesia, mice are able to amble normally around the cage. As an additional precaution, add to the drinking water of the animal Carprofen at 0.0027 mg/ml for 2 days as an analgesic.</li>
<li>Protein expression can be assayed 2-8 days after transfection. However, sustained expression of many proteins has been observed for months.</li>
</ol>
NOTE: All animal procedures were approved by the UCLA Chancellor's Animal Research Committee as mandated by the Animal Welfare Act and the PHS Policy on Humane Care and Use of Laboratory Animals.
Representative Results:
The correct implementation of the in vivo electroporation procedures described above should result in the effective transfection of plasmids in FDB and IO muscles. However, the efficiency of expression of transgenic protein variants will depend on the plasmid, the size and complexity of the protein, the functional properties of the protein, and a number of other variables out of our control. As illustrated in Figure 1 for an FDB muscle electroporated in the presence of a commercial plasmid (pmR-mCherry) encoding for mCherry protein, most of the muscle fibers are transfected with our protocol as illustrated by the fact the most of them display red fluorescence. This does not rule out the possibility that individual fibers exhibit different degrees of protein expression, or that few fibers are not transfected at all 3. It should be noted that the efficient expression of large amounts of fluorescent proteins such as mCherry, EGFP, ECFP, and EYFP, does not impair the muscle fibers’ excitability and excitation-contraction coupling properties. In fact, they are indistinguishable from those in sham transfected muscles (results not shown).
Practical approaches used to verify the localized expression of fluorescently-tagged proteins in skeletal muscle fibers.
The intracellular localization of the di novo expressed transgenic proteins is routinely evaluated by simultaneous acquiring, using the TPLSM, fluorescent images of the respective tags and images of well-identified markers of cellular structures. The most typical of these latter are: a) second harmonic generation (SHG) images, which arise from the myosin anisotropy of the sarcomeric A bands (centered at the M-lines)9,10 and, b) di-8-ANEPPS fluorescence images, which can be obtained by labeling the surface and transverse tubular (T-tubule) system membranes of the muscle fibers with this impermeant potentiometric dye11. In fluorescence images of muscle fibers stained with di-8-ANEPPS, the T-tubules appear as narrow bands of fluorescence oriented approximately orthogonal to the long axis of the fiber. These bands are unequally spaced from each other: they are separated by a long distance which spans across the M-lines, and a short one that spans across the Z-line11.
Expression and localization of α-actinin-EGFP in skeletal muscle fibers.
An example of the expression of a tagged variant of the structural muscle protein α-actinin is shown in Figure 2. This protein is known to be a major component of the Z-line and, as such, is routinely used as a marker of this structure12. We transfected FDB and IO muscles with the plasmid pEGFPN1-α-actinin1 encoding for human (non-muscle) α-actinin tagged at the C terminal with EGFP. Six days after transfection, we found that, as assessed by EGFP fluorescence distribution, α-actinin is mostly expressed at narrow bands equally spaced along the fiber axis. A single band per sarcomere is seen (Figures 2A &amp; 2D). The colocalization of these bands with the Z-lines is demonstrated by comparing the distribution of EGFP fluorescence with the SHG (Figure 2B) and di-8-ANEPPS (Figure 2E) images. As shown by the overlay image (Figure 2C), α-actinin-EGFP bands alternate with the SHG bands, indicating that they are located midway between two consecutive M-bands, coinciding with the location of Z-lines. In transfected muscle fibers stained with di-8-ANEPPS, α-actinin-EGFP bands are seen centered between every pair of T-tubules (Figure 2F) which are known to flank the Z-lines (i.e. separated by a shorter distance), thus indicating that transgenic α-actinin is targeted to the Z-line.
Expression of DHPRα1s tagged at the N-terminus with EGFP
The efficiency of muscle transfection with pEGFPC1.1-DHPR α1s is verified in TPLSM images (e.g. Figure 3A) showing that most fibers express EGFP-DHPRα1s (a transmembrane protein). The most prominent feature of transfected fibers is the double-banded pattern of EGFP fluorescence (Figures 3A &amp; 3B) as would be expected if this protein was targeted to the T-tubules. It can also be observed in Figure 3 that while different fibers display various levels of fluorescence intensity, the banded fluorescence pattern of an individual fiber seems to be maintained homogeneous along the fiber. At higher magnification (Figure 3B), it can be clearly seen that the uneven spacing among bands is similar to that observed in fibers stained di-8-ANEPPS (e.g. Figure 2E). The overlay images (Figure 3B &amp; 3E) illustrate that the SHG bands are located within the larger spacing between EGFP-DHPRα1s bands, corroborating that this protein is at the T-tubules. Additional FRET measurements with the non-fluorescent lipophilic anion DPA- (data not shown) further demonstrate that the EGFP moiety is within few nanometers of the inner leaflet of the T-tubules’ membranes.
Localization and functional evaluation of the expression of EYFP-ClC1
Fibers transfected with pEYFP-ClC1, which encodes an EYFP-tagged construct (at the N-terminal) of the skeletal muscle chloride channel (ClC1), display EYFP fluorescence bands (Figure 4A) with a pattern similar to that observed for EGFP-DHPRα1s’ expression (Figure 3A) and corresponding to the T-tubule arrangement as illustrated with di-8-ANEPPS staining (Figure 2E). As expected, the superimposition of EYFP-ClC1 (Figure 4A) and SHG images (Figure 4B) as shown in the overlay (Figure 4C), illustrate that the SHG bands are centered at the large spacing of between EYFP fluorescence bands.
In order to assess whether the expression of EYFP-ClC1 results in a significant increase in the resting conductance of the muscle fibers, as expected from the overexpression of functional chloride channels, we enzymatically dissociated muscle fibers from the transfected FDB muscle and studied their electrophysiological properties using a two-microelectrodes experimental setup described previously6,11,13. Figure 5 shows results from two fibers: one expressing large amounts of EYFP-ClC1 as assessed from global fluorescence intensity measurements in a standard fluorescence microscope (not shown), and the other is a non-transfected control. The voltage record in Figure 5A (obtained from the muscle fiber expressing EYFP-ClC1) demonstrates that due to the excessive resting conductance, the fiber is almost non-excitable. Current stimulus pulses of up to 400nA (0.5ms) needed to be used in order to elicit a very small active response (no overshoot, Figure 5A). After replacement of the external Tyrode solution to one containing 500 μM of the chloride channel blocker 9-ACA, 200 nA current pulses were sufficient to elicit an action potential (Figure 5B), although much slower and broader than those recorded in the control fiber (Figure 5C). As expected, addition of 9-ACA had minimal effects on this control fiber (Figure 5D).
<img src="/files/ftp_upload/1520/1520fig1tmb.jpg" alt="Muscle tissue diagram with SHG and Di-8-ANEPPS images; shows T-tubule structural analysis." border="0" data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Transfection efficiency of the in-vivo electroporation method. Brightfield (A) and fluorescence (B) images of an FDB muscle transfected with pmR-mCherry. Days after electroporation protocol: 12 days. Please <a href="https://www.jove.com/files/ftp_upload/1520/1520fig1.jpg">click here</a> for a larger version of figure 1.
<img src="/files/ftp_upload/1520/1520figure2.jpg" alt="Microscopy fluorescence, SHG, EGFP, Di-8-ANEPPS; protein dynamics overlay analysis." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. Expression and targeting of α-actinin-EGFP in FDB fibers. Panels A and B are EGFP fluorescence and SHG images, respectively, of a fiber expressing α-actinin-EGFP. Panel C is a superposition of the images in A and B. Panels D and E are fluorescence images of another fiber expressing α-actinin-EGFP and stained with di-8-ANEPPS, respectively. Panel F is the superposition of images in D and E. Days after electroporation protocol: 6 days.
<img src="/files/ftp_upload/1520/1520figure3.jpg" alt="EGFP-α1s-DHPR confocal microscopy with SHG overlay for muscular structure visualization." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3. Expression and targeting of EGFP-DHPRα1s in FDB fibers. Panel A, EGFP fluorescence image of a group of fibers expressing EGFP-DHPRα1s. Panel B is an enlargement of the square in panel A in order to better show the banded pattern of the protein expression. Panel C is the SHG image corresponding to the image in panel A. Panel D is the overlay of the images A and C. Panel E is an enlargement of the area indicated in panel D. Days after electroporation protocol: 20 days. 
<img src="/files/ftp_upload/1520/1520figure4.jpg" alt="Muscle fiber imaging and graph; second harmonic generation, fluorescence microscopy, intensity analysis." data-altupdatedat="1787630408000" data-alt="Figure 4"> Figure 4. Expression and targeting of EYFP-ClC1 in FDB fibers. Panels A and B are EYFP fluorescence and SHG images, respectively, of fibers expressing EYFP-ClC1. Panel C is the superposition of the images in panels A and B. Panel D is an intensity profile measured along the white line highlighted in the image in panel C. Days after electroporation protocol: 7 days.
<img src="/files/ftp_upload/1520/1520figure5.jpg" alt="Transmembrane potential graphs; Tyrode conditions; transfected vs control; electrophysiology study." data-altupdatedat="1787630408000" data-alt="Figure 5"> Figure 5. Electrophysiological assessment fibers expressing transgenic EYFP-ClC1. Panels A and B are voltage records from a fiber expressing EYFP-ClC1 in response to current pulses before and after treatment with 9-ACA, respectively. Panels C and D are voltage records (action potentials) elicited in response to current pulses in a non-transfected fiber before and after treatment with 9-ACA, respectively.

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</ol>

The authors have nothing to disclose.

We describe here the detailed steps that should be followed in order to attain effective transfections of DNA plasmids into skeletal muscle fibers by in vivo electroporation. The main advantages of our approach are the simplicity of implementation, and its minimal invasiveness which results in negligible health hazard to the animals. In reality, the above described electroporation protocols do not entail much more than two subcutaneous injections per foot, followed by an electrical stimulation protocol, all of w...

dna transfection of mammalian skeletal muscles using in vivo electroporation

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.

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

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

Electroporation can be done in vivo to transfect, DNA, plasmids and express transgenically in adult mouse flexor digitorum brevis or FDB and interosseous or IO muscles. The procedure begins with anesthetizing the mouse. The plantar surface of one foot is prepared for the first injection of hyaluronidase by inserting the needle at the heel and guiding it subcutaneously along the foot.After one hour, the animal is re anesthetized and the plasma is injected in the same subcutaneous region of the foot where the hyaluronidase was injected. After 10 minutes, gold-plated acupuncture needles are placed subcutaneously as shown. The electroporation pulses are delivered after connecting the needles to the stimulator if needed.The procedure can be repeated in the contralateral foot. After a period ranging from two to eight days, the animal is sacrificed, the muscles are dissected, and the transfection of the desired protein is verified by observing the fluorescence under a microscope. Hi, I am Marino DeFranco from the laboratory of Julio Vera in the Department of Physiology in the Davy Geffen School of Medicine at UCLA.Today we'll show you a procedure for the Inbivo transfection of transgenic protein expressing plasmid into muscle fibers of adult mice. We use this procedure in our laboratory to study transgenic protein expression and localization, as well as the effect of the protein over expression in muscle fiber physiology. So let's get started.Before starting the in vivo electroporation protocols, mammalian expression plasmids must be amplified to yield concentrations in the range of two to five micrograms of plasmid per microliter of TE buffer. We routinely use Qiagen amplification kits and follow the manufacturers and manufacturer's instructions. We suggest commercial expression plasmids carrying the CMV promoter, for example, EEG FPC one or EEG FPN one from in Vitrogen.As this promoter works very well in skeletal muscle, in vivo transections, we will demonstrate the procedure. Using P EEG FPN one, we have prepared in advance a solution containing two milligrams per milliliter hyaluronidase in sterile tyro. This solution is maintained at room temperature until used to prepare animals.For transfection, we use an anesthetizing box connected to an approved gas anesthetic machine and deeply anesthetize the animal with 4%ISO fluorine in oxygen. Once anesthetized, we apply ointment to protect the eyes of the mouse from dryness during the following anesthesia procedures. Place the animal on a heating pad set at 37 degrees Celsius and maintain the anesthesia using odent face mask.Monitor the anesthetic depth by checking the toe pinch reflex. When the animal is deeply anesthetized. Use a one inch long 33 gauge sterile needle to inject 10 microliters of high UR solution under the skin of the foot pad of one foot of the mouse observing under a dissecting microscope scope, penetrate the skin at a point close to the heel of the foot and advance the needle subcutaneously towards the base of the toes.For approximately a quarter inch, inject the solution gently to prevent too much bulging of the skin. Repeat the procedure with the other foot. If so desired, the injected enzyme will soften over a period of approximately one hour, the connective tissue surrounding the muscle fibers by hydrolyzing hyaluronic acid.This will facilitate the diffusion of the DNA plasmid that will be injected afterwards in the interstitial space between the fibers. After hyaluronidase injection, disconnects the anesthesia and place the mouse in a cage. The animal will recover from anesthesia in three to four minutes.After one hour, anesthetize the animal for a second time. Collect 10 microliters of plasmid solution and place a drop on the back of a clean small whey boat. Pick up the drop with the injection syringe and inject it into the foot pad.Following the same procedure described for the hyaluronidase solution for larger proteins, larger amounts of plasma can be injected. We recommend not injecting more than 20 microliters when more than 15 microliters is necessary. We recommend using tissue glue to close the skin at the needle entry point to prevent any risk of DNA leaking through the puncture point.Disconnect the anesthesia and place the mouse in a cage. Allow it to fully recover from anesthesia and wait for 10 to 15 minutes to prepare for electroporation. Anesthetize the animal as previously done.Select one foot of the animal place one gold plated acupuncture needle or electrode under the skin at the heel, and the second one at the base of the toes. The needles are oriented parallel to each other and perpendicular to the long axis of the foot. The acupuncture needles are 160 micrometers in diameter and 13 millimeters in length.They're sold individually wrapped and sterile. Depending on the needle spacing, adjust the pulse amplitude in order to deliver voltage pulses. Generating an electric field of approximately 100 volts per centimeter.Adjust the pulse duration to 20 milliseconds and the frequency to one hertz. Connect the head of the electrodes to the electrical stimulator using micro clip connectors. Ate the foot muscles by applying 20 electric pulses.Monitor the voltage waveform within oscilloscope. No major contractions are observed in response to the stimuli. If the level of anesthesia is adequate, if so desired while still under anesthesia.Repeat the above electroporation procedure in the other foot after electroporation, return the animal to its cage and maintain it under observation. Until fully recovered from anesthesia, the animal should regain full mobility within five minutes. If there were no adverse reactions and the procedure went normally as an additional precaution, add to the drinking water of the animal carfin at 0.027 milligrams per milliliter for two days.As an analgesic protein expression can be assayed two to eight days after transfection. However, sustained expression of many proteins has been observed for months. The efficiency of transfection of an FDB muscle electroporated with the commercial plasmid P EEG FPN one encoding for the EGFP protein can be readily visualized with UV light coming from either a handheld UV lamp or high intensity UV diodes.Viewing the mouse's foot under the dissecting microscope, it can be observed that most of the muscle fibers are transfected with our protocol as illustrated by the fact that most of them display green fluorescence. The dissecting microscope shows the general transfection pattern of the muscle. Repeating this observation.Using an epi fluorescence microscope allows for the visualization of the pattern of EGFP expression in bundles of muscle fibers or even in individual fibers. The efficiency of the expression of mCherry protein in FDB muscle fibers can be evaluated by contrasting the brightfield and fluorescence images of the muscle. The intracellular localization of the de novo expressed transgenic proteins can be evaluated by using two photon laser scanning microscopy to this end.Images of the fluorescently tagged proteins and images of well-identified markers of cellular structures are acquired simultaneously. The most typical of cellular structural markers are the second harmonic generation or SHG images which arise from the myosin anes atropy of the sarcomeric. A bands centered at the M lines and dye.A annex fluorescence images, which can be obtained by labeling the surface and transverse tubular or t tubule system membranes of the muscle fibers with its imperian potentia metric dye in fluorescence images of muscle fibers stained with dye eight anep. The T tubules appear as narrow bands of fluorescence oriented approximately orthogonal to the long axis of the fiber. These bands are unequally spaced from each other.They are separated by a long distance, which spans across the M lines and a short one that spans across the Z line. An example of the expression of a tagged variant of the structural muscle protein alpha actinin is shown here. This protein is a major component of the Z line and is routinely used as a marker of this structure.We transfected FDB and IO muscles with the plasmid P-E-G-F-P Fpn one alpha actinin, one encoding for human non-muscle alpha actinin tagged at the C terminus with EGFP six days after transfection. We found that alpha actinin is mostly expressed at narrow bands equally spaced along the fiber axis. A single band per sarcomere is seen.The colocalization of these bands with the Z lines is evidenced by comparing the distribution of EGFP fluorescence with either the SHG or the D eight annex images as shown by the overlay image. Alpha Acton and EGFP bands alternate with the SHG bands indicating that they are located midway between two consecutive M bands coinciding with the location of Z lines in transfected muscle fibers stained with d an up Alpha Actinin EGFP bands are seen centered between every pair of T tubules, which are known to flank the Z lines. This shows that transgenic alpha actinin is targeted to the Z line.The expression and targeting of the N terminal EGFP tagged DHPR alpha one s is shown in figure three.EGFP. Fluorescence is mostly seen as pairs of bands spaced with a pattern similar to that of the T two Es.Super imposing SHG and E-G-F-P-D-H-P-R Alpha one s images demonstrates that there are two EGFP fluorescent bands between every two consecutive SHG bands, suggesting that DHPR alpha one s has been correctly targeted to the T two ES D eight and abstaining, not shown, provides a definite proof of DHPR Alpha one St.Tubule targeting fibers transfected with P-E-Y-F-P CLC one, which encodes an EYFP tagged construct at the end. Terminus of the skeletal muscle chloride channel CLC one display EYFP fluorescence spans and correspond to the T tubule arrangement as illustrated with D eight and abstaining.As expected. The super imposition of EYFP CLC one and SG images as shown in the overlay illustrate that the SHG bands are centered at the large spacing between EYFP fluorescence bands. In order to assess whether the expression of EY FP CLC one results in a significant increase in the resting conductance of the muscle fibers as expected from the overexpression of functional chloride channels, we enzymatically dissociated muscle fibers from the TRANSFECTED FDB muscle and study their electrophysiological properties using a two micro electrodes experimental setup.This image shows results from two fibers. One expressing large amounts of EYFP clc. One has assessed from global fluorescence intensity measurements in a standard fluorescence microscope, and the other is a non transfected control.The voltage record was obtained from the muscle fiber expressing EY FP CLC one. A current pulse larger than that needed to excite a control. Fiber elicits only a small regenerative response in the transfected fiber when the external tyro solution was replaced with a solution containing 500 micromolar of the chloride channel blocker nine Racine carboxylic acid or nine A CA.A smaller pulse elicited inaction potential, although much slower and broader than those recorded in the control fiber as expected.Addition of nine a CA had minimal effects on the control fiber. We conclude that the fiber expressing EY FP CLC one is almost non excitable due to the excessive resting chloride conductance. We have just shown you how to transfect transgenic protein expressing plasmid into live skeletal muscle fiber using electroporation, and also how to evaluate the transfection efficiency and the localization and function of the transfected proteins.So that's it. Thanks for watching and good luck with your experiments.I.

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Research

JoVE Journal

Biology

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described.  This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

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.

DNA Electroporation, Isolation and Imaging of Myofibers

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

Learn in vivo electroporation for DNA transfection into mouse skeletal muscles using hyaluronidase and gold-plated needles. This method enables efficient gene delivery and fluorescence visualization for muscle biology research.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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