Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

This protocol is significant because it allows us to study the function of proteins, such as extracellular matrix proteins, that are up-regulated in later stages of myotube formation or maturation. This method can be used to knock down any gene of interest in C2C12 cells by using specific shRNAs and the respective analytical tools for phenotyping. The main advantage of this method is that we have generated C2C12 cells that can continuously suppress our genes of interest, even in mature myotubes.

The most important aspect of this procedure is to maintain C2C12 cells in the undifferentiated state, which means at a low cell density during routine cell culture. The day before transfection, seed five times 10 to the fourth C2C12 cells per milliliter in two milliliters of complete DMEM per well in a six-well plate to achieve a 40 to 50%confluence after overnight incubation at 37 degrees Celsius and 5%CO2. The next morning, combine 100 microliters of 25-millimolar sodium chloride in one 1.5-milliliter reaction tube per culture well with 25.5 microliters PEI stock solution of an undifferentiated C2C12 cell culture for a five-minute incubation at 37 degrees Celsius.

Next, combine three micrograms of plasmid DNA with 100 microliters of 25-millimolar sodium chloride per culture well for a five-minute incubation at 37 degrees Celsius. At the end of the incubations, combine the entire volume of each diluted PEI reagent with each diluted plasmid solution with gentle pipetting for a 25-minute incubation at 37 degrees Celsius. During the incubation, replace the supernatants of the C2C12 cell cultures with DMEM without antibiotics or serum.

At the end of the incubation, add the entire volume of each transfection mix to each well in droplets, while continuously moving the cell culture dish. After six hours in the cell culture incubator, replace the medium in each well with complete DMEM. 24 hours after the transfection, replace the complete DMEM in each well with selection medium supplemented with five micrograms per milliliter of puromycin, and return the cells to the cell culture incubator for 10 to 14 days or until stable C2C12 cells are observed.

Then expand the puromycin-resistant C2C12 cells in low cell density cultures in the presence of five micrograms per milliliter of puromycin, and cryopreserve six to 10 vials of cells for future use. To prepare the C2C12 cells for differentiation, seed 1.5 times 10 to the fifth stable puromycin-resistant C2C12 cells in one milliliter of selection medium per well in a 12-well plate, and incubate at 37 degrees Celsius and 5%carbon dioxide until the cultures are 95%confluent. To induce differentiation, replace the medium in each well with serum-free DMEM every two days following the myotube formation on an inverted bright-field microscope equipped with a camera.

For myosin heavy chain immunostaining of the differentiated cells, seed five times 10 to the fourth puromycin-resistant C2C12 cells in 500 microliters of complete DMEM per chamber onto an eight-well chamber slide, and return the cells to the cell culture incubator. When the cells reach 95%confluency, replace the selection medium in each well with 500 microliters of serum-free DMEM. At the appropriate experimental time points, rinse the differentiating cells in each well three times with 5 milliliters of PBS per wash before fixing the cells with 200 microliters of 4%paraformaldehyde in PBS per well.

After 15 minutes, wash the cells three times with fresh PBS per wash as just demonstrated followed by incubation with 200 microliters of 5-molar glycine in PBS per well for five minutes. At the end of the incubation, wash the cells three times before permeabilizing the cells with 200 microliters of 1%non-ionic surfactant in PBS for 10 minutes. Next, block the nonspecific antibody binding sites with 200 microliters of 5%bovine serum albumin in PBS per well for one hour followed by three washes in PBS.

After the last wash, label the cells with 200 microliters of myosin heavy chain antibody for two hours at room temperature. After three PBS washes, incubate the cells with the appropriate secondary antibody for two hours at room temperature, protected from light. After three final washes, aspirate the PBS, and mount the cells with one drop of mounting medium supplemented with DAPI and a coverslip.

Then observe each chamber on a fluorescence microscope using the appropriate filter set. To assess the knockdown efficiency in the cell cultures by western blotting, first seed three times 10 to the fifth puromycin-resistant C2C12 cells in two milliliters of complete DMEM per well in a six-well plate. After 24 hours, rinse the cultures with PBS, and initiate differentiation of the cells with two milliliters of serum-free DMEM as demonstrated.

For analysis of the secreted proteins at the appropriate experimental time points, collect one milliliter of serum-free conditioned medium from each well into individual 1.5-milliliter reaction tubes for centrifugation. After centrifugation, transfer one milliliter of the conditioned medium supernatants into new 1.5-milliliter reaction tubes, and precipitate the proteins with 391 microliters of trichloroacetic acid in non-ionic surfactant per tube. After 30 seconds of vortexing, incubate the mixtures on ice for 10 minutes.

At the end of the incubation, pellet the precipitated proteins by centrifugation, and wash the protein pellets three times with fresh ice-cold acetone and one centrifugation per wash. After the last wash, air-dry the pellets for three to four minutes at room temperature before resuspension in 50 microliters of SDS-PAGE sample buffer per tube. Then boil the samples for five minutes at 95 degrees Celsius before western blot analysis according to standard protocols.

For intracellular and membrane-bound protein analysis, rinse the cell layer in each well with two milliliters of PBS per well, and use a cell scraper to carefully dislodge the cells in one-milliliter volumes of PBS. Transfer the cells into individual 1.5-milliliter tubes for centrifugation followed by three washes in one milliliter of PBS per tube per wash via centrifugation. After the last wash, resuspend the cell pellets in 200 microliters of lysis buffer supplemented with EDTA-free protease inhibitor cocktail reagent for a 30-minute incubation on ice.

At the end of the incubation, ultrasonicate the samples for 15 seconds on ice with a power output setting of 10 at an operating frequency of 23 kilohertz, and remove the cell debris by centrifugation. Transfer the supernatants into new 1.5-milliliter reaction tubes, and determine the protein concentrations according to standard protocols. Combine 100 micrograms of protein with 5X SDS-PAGE sample buffer in a total volume of 60 microliters per sample, and boil the samples for five minutes at 95 degrees Celsius.

Then analyze the samples by western blotting for the presence of the desired target protein. The selection of puromycin-resistant C2C12 cells can be achieved within 10 to 14 days of transfection due to an efficient elimination of the non-resistant cells. Typically, more than 80%of the C2C12 cells detach from the cell culture dish during this time period and are removed during routine cell maintenance.

Puromycin-resistant C2C12 cells expressing the control or the ADAMTSL2 shRNA retain their spindle-shaped, elongated cell morphology at a low cell density, as well as their ability to differentiate into myotubes. C2C12 differentiation upon serum withdrawal can be monitored by bright-field microscopy and immunostaining for the myotube marker myosin heavy chain. Myosin heavy chain-positive myotubes are observed between three to five days after differentiation initiation and are multinucleated, as observed by the presence of more than one DAPI-positive nucleus within the cell boundaries.

As the gene expression data in proliferation conditions demonstrate, the knockdown efficiency of the transfection is 40 to 60%Western blot analysis can be performed to confirm the success of the knockdown in cell lysates obtained, for example, from C2C12 cells stably expressing shRNA that targets ADAMTSL2 compared to control shRNA expressing cells. Be sure to maintain the puromycin concentration during the selection process high enough to eliminate all of the non-transfected C2C12 cells, or the non-transfected cells may outgrow the transfected cells. This assay allows us to determine the function of extracellular matrix proteins that are expressed later in muscle development, even if they are induced five or 10 days after C2C12 differentiation.

Remember that the RNA extraction reagent and beta-mercaptoethanol should be handled in a fume hood and to handle the trichloroacetic acid according to the appropriate safety protocols. Thanks for watching, and good luck with your experiment.