The overall goal of these myoblast maintenance, transfection, and transduction procedures is to establish a myotonic dystrophy one cell model for the study of myotonic dystrophy and other muscular diseases. This method can help establish the M1 myoblast cell models that will be useful in investigating the detailed mechanism of the M1 pathogenesis. This multi tiered gene manipulation of C2C12 myoblasts enables the modeling of sequential and genetic pathological events in the M1.24 hours before their transfection plate seven times 10 to the fifth C2C12 cells per well in a six well plate.
The next day add two micro grams of the plasmid DNA of interest into 200 micro liters of room temperature, serum free, medium and briefly vortex the solution. Next, add six micro liters of room temperature transfection reagent to the medium, and immediately vortex the solution for 30 seconds. Then incubate the medium at room temperature for 30 minutes.
During the last five minutes of the incubation. Replace the medium in each well with two point five milliliters of fresh serum free growth medium, and add 200 micro liters of transfection mixture to each well. Return the plate to the cell culture incubator for four hours.
Then check the cells under a microscope for changes in their morphology and attachment. If cytotoxicity is observed, replace the serum free medium with growth medium. 48 hours after transfection, add the appropriate selection antibiotic in fresh growth medium every two days for 10 days.
Until the control transfected without plasmids well does not contain live cells. To prepare the retrovirus, first plate four times 10 to the sixth ecotropic HEK293 based packaging cells in a 100 millimeter plate containing cell culture medium. The next day, mix 15 micro grams of the plasmid DNA of interest in one milliliter of serum free growth medium, and briefly vortex the tube.
Next, add 30 micro liters of room temperature transfection reagent to the medium, and immediately vortex the solution for 30 seconds. After 30 minutes at room temperature, add the transfection mixture drop wise to the ecotropic cells, and gently swirl the plate. Return the plate to the cell culture incubator for 24 hours, replacing the medium with 10 milliliters of 37 degree celsius fresh growth medium the next day.
48 hours after the transfection, use a pipet to transfer the retro viral containing supernatants into a single 50 milliliter conical tube for overnight storage at four degrees Celsius. Then add 10 milliliters of new warm medium down the side of the plate, and return the culture to the incubator. Harvest the second batch of retro virus 60 hours after the transfection.
Pooling it with the retrovirus collected at 48 hours. Spin down the supernatant to remove any cellular debris, and transfer the retrovirus into a new 50 milliliter tube. Now wash each well of a C2C12 cell culture with three milliliters of room temperature PPS followed by the addition of 500 micro liters of zero point two five percent trypsin EDTA for a five minute incubation at 37 degrees Celsius.
When the cells have detached, neutralize the reaction in each well with three milliliters of growth medium and triturate the culture six to eight times to disassociate the cells into a single cell suspension. After counting add eight times 10 to the fifth cells to a 60 milliliter plate, and add three milliliters of growth medium. Then infect the cells with zero point five milliliters of retro virus and return the plate to the incubator for 48 hours.
After two days, feed the cells with three milliliters of fresh growth medium, supplemented with the appropriate selection antibiotic everyday for five days. Until the untransduced C2C12 control culture contains no more living cells. For C2C12 cell lentiviral transduction, in a bio-safety level two cabinet, plate four times 10 to the sixth 293T cells in a 100 millimeter plate with HEK293 cell culture medium.
The next day, mix the lenitviral packaging vectors with eight micro grams of the appropriate lenitviral transfer vector, in one milliliter of serum free cell culture medium. Vortex the mixture briefly and add 36 micro liters of pre-warmed transfection reagent. Then vortex the medium for another 30 seconds, and incubate the mixture at room temperature for 30 minutes.
At the end of the incubation, add the transfection mixture to the 293T cells, gently swirl the plate, and return the cells to the incubator. After 24 hours, replace the transfection mixture with 10 milliliters of fresh culture medium. After 48 hours, transfer the lentivirus containing supernatant into a 50 milliliter conical tube for four degree celsius storage, and add 10 milliliters of fresh, warm medium to the plate.
After 60 hours, pool the second batch of supernatant with the lentivirus collected at 48 hours, and centrifuge the virus to remove any cell debris. Then add zero point five milliliters of lentivirus to a 30 to 40 percent confluent culture of C2C12 CUG200 cells seated in a 60 millimeter plate as just demonstrated. 72 hours after transfection, replace the lentivirus supernatant with fresh medium, supplemented with the appropriate selection antibiotic.
Refreshing the culture medium every day for five days with fresh selection antibiotic supplemented medium, until all of the untransduced C2C12 CUG200 control cells are dead. After drug resistance selection, the GFP expression and myotube formation of differentiated myobalsts can be assessed by a manofluorescent analysis. Quantification of the myotube formation in this representative experiment demonstrates that the fusion indecies and myotube areas are decreases significantly by abnormal CUG expansion and GFP CUG 200 transfected cells.
Celf1 upregulation is also associated with CUG expansion in myogenic dystrophy one. Further, western blotting consistently demonstrates and elevation of Celf1 protein levels with CUG inhibited expression of MyoD, MyoG, and Mef2C during differentiation. Suggesting that CUG expansion induces myotube defects and impaired myoblast differentiation.
Western blot analysis also reveals both that FLAG-tagged Celf1 is present only in retroviral vector transduced cultures, and that it's expression is up regulated in these cultures. Indeed myotube formation is rare in Celf1 over-expressing cells compared to control cultures. Suggesting that the over-expression of Celf1 severely impairs myoblast differentiation.
Further, the level of endogenous Celf1 protein and double antibiotic selected lentiviral vector transduced GFP CUG 200 cells is markedly reduced in the presence of Celf1 short hairpin RNA. With the rescue of myotube formation, myotube fusion indecies and myotube areas observed in these cultures. Meanwhile, real time RTPCR results suggest that the expression of MyoD, MyoG, and Mef2C are significantly increased in Celf1 short hairpin RNA expressing cells supporting the possibility of a Celf1 knock down rescued myocyte differentiation deficiency in these cultures.
While attempting this procedure, it's important to monitor the C2C12 cell density to be below 70 percent confluence. Following the cell transfection, treat the cells with serum free medium for at least four hours, but no more than 12 hours. While monitoring the cells cytotoxicity.
Through the use of plasmid transfection, retroviral and lentiviral transduction, we have successfully established a myotonic dystrophy cell model which will benefit research in this and other muscular diseases.
