使用して哺乳類の骨格筋のDNAトランスフェクションインビボでのエレクトロポレーション

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.