The overall aim of this procedure is to non-invasively evaluate the success of myoblast transplantation using a reporter gene approach and bioluminescence imaging. This is accomplished by first introducing the tri fusion reported gene that expresses f Luke, MRFP and SR three 90 tk under the control of the CMV promoter into the immortalized C two C 12 myoblast cell line. The second step is to quantify the number of myoblasts that express the porter gene.
In this study, transfection efficiency is calculated by counting MRFP expressing cells on a fluorescence microscope. Next 10 to the five engineered cells in a volume equal to or less than 15 microliters are implanted intramuscularly into the hind limb of dystrophic MDX mice. UNT transfected cells are implanted into the contralateral hind limb as a control, then a baseline scan is performed.
The final step is to intraperitoneal, inject the f Luke reported gene substrate Lucifer following an uptake period of around 20 minutes, conduct a second bioluminescence scan to acquire an image of implanted cells. Ultimately, uptake of Lucifer is observed specifically in f Luke expressing cells with no detectable bioluminescence in control cells that don't express the report gene. This method could help answer key questions in the field of mild loss transfer technology such as the migration of cells post implantation, as well as cell survival following transplantation.
The implications of this technique extend towards therapy for muscular diseases such as Duchenne muscular dystrophy because it's a fast, reliable means of non-invasively targeting cells Following transplantation, we can thus assess the engrafted area over time and evaluate the success or failure of the treatment. Demonstrating the procedure will be Kelly gut pe, a graduate student, and Rebecca Mcg, a research technician from our laboratory, sorry, Culture C two C 12 myoblasts in high glucose dcos modified eagles medium with 10%fetal bovine serum according to standard procedures for this cell line. Do not allow the cells to become more than 80%confluent as this will deplete the myotic population.
To prepare the cells for transfection first, harvest the cells from the stock flask by briefly covering the HBSS rinse cell layer with trypsin. Then quickly aspirating the trypsin and placing the flask in the tissue culture incubator for five minutes. During the incubation, prepare two T 75 flasks each containing nine milliliters of culture medium.
When the incubation time is complete, add 10 milliliters of media to the flask and rinse the media over the bottom of the flask four to five times to ensure collection of all cells. Then seed each of the T 75 flasks with one 10th of the cell suspension from the stock T 75 flask. When first attempting this procedure, conduct an MTT assay to assess the effect of cell labeling on cell survival and differentiation and the viability of engineered myoblasts prior to transplantation.
Once the cells have reached 50%confluence, transfect one T 75 flask with 36 micrograms of the CMV Tri fusion reporter gene in a 4.5 milliliter volume of optimum using Lipofectamine 2000 according to the manufacturer's instructions. In addition, perform a mock transfection of the second T 75 flask using only lipo 2000 and optimum without DNA as a negative control. Allow the cells to transfect for at least 20 hours at 37 degrees Celsius the next day, aspirate the transfection medium and replace with the suitable volume of culture Media using an inverted fluorescent microscope.
Capture both brightfield and red fluorescence images to calculate transfection efficiency. After harvesting the transfected myoblast by trypsin and resus suspending the cells in four milliliters of complete medium, use a hemo cytometer to perform a cell count. Pipette 10 to the six cells into a sterile 1.5 milliliter micro tube with a transfection efficiency of around 10%This number of cells should give rise to 100, 000 detectable Lucifer arrays expressing cells after transplant, the final injection volume should be 15 microliters.
If the volume in the tube is greater than this, then centrifuge the micro tubes at 2000 RPM for one minute, asate the supinate, and then reus bend in a maximum volume of 15 microliters of growth. Medium lacking FBS After inducing anesthesia in the mouse using 2%isof fluorine switch to 1.5%isof fluorine for anesthesia maintenance. During the procedure, place the mouse in a prone position and pluck the hair from the dorsal hind limb area.
Ensure that the C two C 12 myoblasts are well suspended, and then load the cell suspension into an insulin syringe. Inject the cells directly into the lateral head of the gastro muscle at a 30 degree angle. After injecting transfected myoblasts into the right hind limb, repeat the procedure with UNT transfected myoblasts in the contralateral hind limb.
Perform a baseline bioluminescence scan. One person should quickly transfer the mouse to the stage in the optical scanner laying the mouse in a prone position. A second person simultaneously hooks up the anesthetic line to ensure the mouse remains anesthetized.
Gently extend the hind limbs and use medical tape to hold the limb in place so that both areas of injection are visible. Close the chamber and ensure that no light can access the interior of the scanner as this will increase the background signal detected. The scanner should be set to parameters included in the manufacturer's instructions for bioluminescence imaging.
Draw a region of interest around the plucked injected area and start the scan to obtain the baseline image while the mouse is anesthetized.Intraperitoneal. Inject 150 milligrams per kilogram of firefly luciferase substrate Lucifer in. Place the mouse into a clean cage and allow it to recover from the anesthesia.
Allow a 15 minute uptake period before preparing the mouse for the next scan. Following the 15 minute uptake period. Anesthetize the mouse and transfer to the scanner as before.
Perform a second by luminescence scan in the same manner as the background scan. The total 20 minute uptake period should provide maximal signal intensity, but a subsequent scan may be performed if desired. This schematic shows the CMV Tri Fusion reporter construct.
The left panel shows the bright field image of C two C 12 myoblasts transfected with the fusion reporter plasmid, and the right panel shows the same region as a fluorescence image as seen here. A region of interest is drawn to enclose the plucked hind limb area where myoblasts were injected. Bioluminescence is not detected during a background scan.
At 23 minutes after injection of Luciferian, a clear signal is detected from the right hind limb where Luciferase expressing myoblasts were injected. No bioluminescence is detected in the contralateral hind limb injected with unresected myoblasts as seen. Here immunohistochemistry using a firefly luciferase antibody confirms intramuscular implantation of transfected C two C 12 cells.
While attempting this procedure, it's important to remember to use aseptic tissue culture techniques and appropriate animal handling practices after its development. This technique paved the way for researchers in the field of muscular diseases to explore the efficacy of cell therapy for now in preclinical small animal models of a disease. Then later in clinical setting in patients, after watching this video, you should have a good understanding of how to implant cells and how to non-invasively localize engineered transplanted myoblasts using bio luminescence imaging.
