The overall goal of this procedure is to identify and track cell cell fusion events in vivo. This is accomplished by first transecting, a flocked luciferase gene in donor cells. The second step of the procedure is to deliver transfected cells to tissues of a mouse genetically modified to Constitutively express Cree recombinase.
The third step is to image luminescence in the tissues of recipient mice, which corresponds to cell cell fusion events. This is followed by analysis of signal intensities according to the experimental conditions. Ultimately, results can show localization and trafficking of cell cell fusion products in living mice through the detection of inducible luminescence associated with cell fusion.
The method can help address key questions in the fields of development, stem cell biology and cancer biology, such as whether cell fusion facilitates stem cell programming, or whether cancer metastasis relies on cell fusion. Demonstrating the procedure will be Anthony Springers, Nicholas Co, and Brian Freeman, students in our laboratory and gal queen song, an associate scientist in the department of medicine Begin with trypsin cells for in vivo fusing when they are 70 to 80%confluent. In this case, mesenchymal stem cells are employed, inactivate the trypsin with an antibiotic free alpha, MEM, complete medium, and spin down the cells carefully aspirate the supernatant and resus suspend the cell pellet in one milliliter of one XPBS Once the cells have been counted, using a hemo cytometer transfer 1.5 million cells to a centrifuge tube and pellet those cells.
After carefully aspirating the supernatant Resus. Suspend the pellet in 300 microliters of R buffer containing six micrograms of plasmid following a manufacturer's protocol. Place three milliliters of EBU into the electroporation docking port.
Then transfer 100 microliters of the cell plasmid solution to a neon pipette tip. Position the pipette tip into the electroporation device and electro purate. The cells.
Now transfer those cells into a 15 milliliter conical tube containing 9.7 milliliters alpha MEM, complete medium. Repeat the electroporation process twice to yield a total of 300 microliters electroporated cells, all pooled in the alpha MEM complete medium. Then add the cells to a T 1 75 flask containing 10 more milliliters of alpha MEM Complete.
Replace the medium 24 hours following the transfection cell survival after electroporation is about 30%which yields about half a million viable cells per flask. After two or three days, the cells will be 70 to 80%confluent. Then transfer and count the cells just prior to injection Resus.
Suspend the cells in alpha MEM complete at a concentration of 1 million cells per 50 microliters medium, and injecting them as soon as possible to minimize cell death. This procedure utilizes mice engineered to express Cree recombinase ubiquitously after anesthetizing a mouse using isoflurane before proceeding. Ensure that it is at a surgical plane of anesthesia using a pain test.
Now remove hair from chest region using hair clippers or a chemical hair remover. Further, prepare the mouse for surgery by intubating it with an 18 gauge catheter, placing it on a mouse ventilator and warming pad. Now perform a thoracotomy with a lateral incision across the fourth intercostal space.
Obtain a clear viewing angle of the heart. Bend the tip of a 28 gauge needle attached to a one milliliter syringe to nearly 90 degrees. Load the syringe with 50 microliters of the prepared cells, including some additional air so the entire cell volume can be ejected from the needle.
Now make 2 25 microliter intra myocardial injections of the transfected cell suspension into the anterior space of the left ventricle. It is important to be sure the cell injection is in the tissue. This can be confirmed by noticing the tissue branching while the injection is done Following the injections.
Use absorbable sutures like Vicryl sutures to close the ribs and muscle layers. Complete the surgery by closing the skin using four aught nylon or silk. When performing the surgery and injection, move swiftly as success hinges on the brevity of this procedure.
Now allow the mouse to recover from anesthesia on a warming plate and extubate the mouse when it coughs or exhibits reflex activity. For controls, cream ice should be injected with alpha MEM, medium, not containing transfected cells and wild type mice should be injected with the transfected cells. Anesthetize the mice to be imaged using 4%isof fluorine.
Prepare the mice with an intraperitoneal injection of D Lucifer. Place each anesthetized and luciferian loaded mouse supine. In the imaging box, several mice can be imaged concurrently, including a sham injection control mouse.
Using a gas mask, maintain their anesthesia with one to 2%isoflurane. Position the imaging box under the microscope and open a commercially available software package to handle the imaging. Prepare the software by first setting the appropriate exposure time.
Next, set the shape of the area to image on one mouse and use that same imaging shape for every other imaged mouse. Then set the subject height at 4.5 centimeters. Acquire a luminescence intensity signal corresponding to the mouse or mice within the field of view.
Then save the unmodified image file. Process the images to remove the background signal corresponding to the control mouse intensity values above the background luminescence correspond to fused cells within the animal. We find image J open source software to be easily adapted and reliable for image post-processing.
To conduct this analysis, it is most crucial that the images obtained from the Xeno gen software be exported as T files. Intensity analysis can be conducted using commercially available or open source image analysis software. Typically, a region of interest is selected to compare intensity data between animals and experiments to determine the sensitivity of the imaging system.
A cell line that Constitutively expresses luciferase was injected into the myocardium of C 57 black six mice at varying concentrations. Six hours later, the mice were imaged for luminescence signal. A signal was detected in two of six mice injected with 1000 cells, and in all six animals injected with 10, 000 cells.
After watching this video, you should have a good understanding of how to identify and track cell cell fusion products in vivo through detection of inducible luminescence.
