DNA Electroporation, Isolation and Imaging of Myofibers

The overall goal of this procedure is to isolate myo fibers from the flexo digitorum brevis of mice, so the sarcolemma can be subsequently damaged through laser wounding. The main advantage of this technique is that protein function and localization can be studied in mature myo fibers without having to generate transgenic mice. This method can be adapted to test pharmaceutical agents and other methods of cell wounding.

Begin with thawing out the collagenase solution. Next, prepare two wells from a 12 well plate. For each fiber to be isolated in one, well add a milliliter of prewarm, DMEM plus BSA load the other well with a milliliter of Prewarm one x ringers.

Also prepare a 35 millimeter cigar dish with 10 milliliters of fresh one x ringers. After removing the feet from the transfected animal pin one foot tight sole facing up onto the sigar dish with 30 gauge needles through each of the five toe tips. And at the ankle, prepare each foot as follows.

First, snip the skin. Start at the ankle and cut along the hairline to the toes. Be careful to not nick the muscles beneath the skin.

Next, grip the skin at the base of the ankle and cut across the heel. Then lift the skin flap using forceps, and point the scissors toward the skin, not at the muscle, and carefully snip the connective tissue to remove the skin. Now to remove the FDB muscle, begin with cutting the large tendon at the heel to free it from the bone.

Then dissect the FDB bundle from the large white tendon. Carefully remove the attached connective tissue in this process. Once completed, lift the bundle off the underlying tendons to reveal bright white tendons leading to the digits.

The bundle should lift up easily. Then free the bundle from the foot by cutting the white tendon near the toes. Transfer the muscle bundle to the well of DMEM plus BSA, and repeat the process on the other foot before proceeding.

It is possible to now check the success of the electroporation, which is covered in the text protocol. Perform this diagnostic check on an inverted fluorescent microscope. If successful, the transfection efficiency can be up to 90%After all the FDB bundles have been isolated, add 100 microliters of collagenase solution to each.

Well incubate the plate in a humidified 37 degrees Celsius incubator with an atmosphere of 10%carbon dioxide for about an hour. This will vary between disease models and also depends on the enzymes efficiency. Once the incubation is completed, carefully transfer the FDB bundles to the ringer solution.

Wells then tritrate the bundles using a one milliliter pipette with a widened bore, do this gently about 15 times during the iteration, observe that the intact fibers fall off into the solution. If this is not occurring, then the incubation in collagenase needs to be extended. Proper attention to the digestion.

Time and force of titration are critical in this procedure. Over digestion, fiber fragmentation, and sarco leal damage can occur leading to a suboptimal muscle preparation. Now collect the isolated fibers in the solution and transfer up to half a milliliter of solution to the culture plate.

Continue to let the muscles digest for another 15 minutes. Then repeat the tation and collect the isolated fibers again if needed. Wait another 15 minutes and repeat the process for a third time.

After the fibers have been allowed to attach to the dish for at least 15 minutes, dilute the residual collagenase with fresh ringers or change the solution altogether. The fibers are now ready for imaging plasmid. DNA coding for fluorescently tagged protein was transfected into the FDB muscle bundle.

The CMV promoter is well suited for this purpose. Fluorescence could be seen in several isolated myo fibers, seven days post electroporation. Then the FDB muscle was digested and myo fibers were isolated in this process.

Damage to the myo fibers can occur as indicated by the white arrow. The black arrows show the blabbing in the sarcolemma membrane. Such fibers were removed from the experiment.

Uninjured myo fibers were imaged using DIC fm. Dye number 4 64 was present minimal FM 4 64 fluorescence at the myofiber membrane prior to injury. Viewing the FM loaded myo fibers by confocal microscopy revealed greater detail.

The centered myofiber is devoid of nuclei. Such nuclei can influence FM dye analysis and should be avoided to induce membrane damage. Place the ROI cross hair on the FM D positive sarcoma begin the membrane ablation protocol Once mastered.

This technique can be completed in approximately two hours. And while attempting this procedure, it's important to remember that digestion time is influenced by collagenase activity, genetic background, and type of disease Model.