This protocol provides a workflow for the production of infectious adenoviral particles that are used to transduce a gene of interest into naive T cells, which upon atopic expression of the transgene or differentiated into regulatory T cells or tregs. First the gene of interest is cloned into the adenoviral destination vector. The vector is then transferred into the adenovirus producer cell line, HC 2 93 A and infectious adenoviral particles are generated.
The resulting viruses are used to infect naive T cells from mice with an adenovirus receptor transgene after infection. The cells are then rested to ensure expression of the gene of interest. In naive T cells, the T cells are activated in polarizing conditions to induce treg differentiation.
Then the cells are fixed and stained for flow cytometric analysis. Ultimately, results can be obtained that show expression of the treg differentiation marker, Fox P three and the infection marker GFP, which may be influenced by the overexpressed gene of interest. The main advantage of this technique over existing methods like retroviral gene transduction is that it allows overexpression of a gene of interest in naive T-cells.
This results in a high level of overexpression present at the moment of T-cell activation. This method can help answer key questions in the regulatory T-cell field. For example, about the post transcription gene regulation that underlies the establishment of T-Rex cell identity.
Begin this protocol by cloning the gene of interest into an entry vector. The PCR amplified gene of interest is blunt and ligated into the topoisomerase coupled vector P enter D topo. The gene of interest is then transferred by LR Recombination into the P kakadu destination vector.
It contains the adenoviral genes and an internal ribosomal entry site or iris, followed by the open reading frame of GFP to create an adenoviral expression vector with an infection marker. Next 10 micrograms of the adenoviral expression vector is linearized. In a PAC one restriction digest, the DNA is then precipitated and resuspended in water at a concentration of three micrograms per 100 microliters.
Linearization liberates the viral inverted repeats or ITR, which are required for replication and encapsulation of the viral DNA into virus particles. All of the following steps should be performed under the appropriate biosafety conditions for adenovirus infection. To prepare linearized vector for cot transfection transfer six microliters of jet PEI reagent into 94 microliters of 50 micromolar sodium chloride vortex.
Briefly then, while vortexing add the mixed solution to 100 microliters of linearized adenovirus vector. Incubate this transfection mix for 15 to 30 minutes at room temperature. Next, remove HEC 2 9 3 A cells grown to 50%co fluency on six well plates from the incubator.
Dispense the solution dropwise into the wells containing HEC 2 9 3 A cells and incubate them at 37 degrees Celsius and 10%carbon dioxide. Add 0.5 milliliters of fresh medium every three days using GFP expression as an infection marker. Evaluate the transfection efficiency visually with an inverted fluorescence microscope every two to three days check for cytopathic effects evidenced by areas with enlarged and rounded cells that start to detach.
This is indicative of efficient virus generation. Once broader zones of cytopathic effects appear, all cells will be infected within 24 to 72 hours. When all of the cells show signs of cytopathic effects, but before an overall detachment of cells occurs, harvest the cells by gentle pipetting and transfer them with the supernatant to a 15 milliliter polystyrene tube.
Next to rupture the cells freeze the suspensions on dry ice for 15 to 20 minutes, then quickly thaw them at 37 degrees Celsius. Repeat this, freeze and thaw cycle two more times. The ruptured cell suspension is now referred to as the primary virus.Lysate.
Keep all virus lysates on ice. If they'll be used within a day for long-term storage, freeze them at negative 80 degrees Celsius. Keep in mind that any additional freeze thaw cycles will reduce the virus tighter by 30 to 50%To amplify the primary virus lysate infect HC 2 93 A cells grown to 90%confluence on 15 tissue culture dishes with half of the primary virus, lysate and incubate for at least 36 hours.
After 36 hours check for infection by fluorescence microscopy, nearly all cells should be infected. Once all of the cells show cytopathic effects but not detachment, harvest the cells by gentle pipetting with efficient virus production. This state is reached within 48 hours of infection, transfer the cells and supernatant to a 50 milliliter polystyrene tube.
Spin cells down at 300 G for 10 minutes at four degrees Celsius. Following the spin, remove the supernatant and resuspend the pellet in a suitable volume of medium. One milliliter should result in a titer in the range of 10 to the 10th per milliliter.
Perform three freeze thaw cycles as before to disrupt the cells. Then centrifuge at 900 G for 20 minutes at four degrees Celsius. After the spin, carefully transfer the supernatant, which is now referred to as the concentrated virus, lysate to a cryo tube.
Make aliquots for multiple infections and store them at negative 80 degrees Celsius. Finally, determine the virus titer by fax analysis of infection marker expression in a 5 4 9 cells. According to the instructions in the accompanying document, this part of the protocol is performed using naive rusting CD four T cells isolated from D 1110 transgenic car delta one transgenic mice for small scale experiments.
Peppe an appropriate volume of viral lysate to achieve an MOI of 50 into one well of a 96 well round bottom plate. Add up to four times 10 to the fifth T cells in a final infection. Volume of 50 microliters in T-cell.
Medium medium. This setup allows parallel experimental approaches. Incubate cells for 90 minutes at 37 degrees Celsius in a 5%carbon dioxide incubator.
Following the incubation, spin the cells down at 300 G for five minutes at room temperature after the spin resus, suspend them in 200 microliters of PBS centrifuge again and take off the supernatant Resus suspend cells in 200 microliters of T-cell medium without stimulating antibodies and without IL two or other cytokines. Then rest the cells for 40 hours at 37 degrees Celsius in a 5%carbon dioxide incubator to allow expression of the gene of interest before activation to activate and polarize the T cells pipette anti CD three and anti CD 28 coupled beads into a small reagent cup. The number of beads should be equal to the number of cells, then tenfold the volume of PBS and place them on a magnet for two minutes.
Next, take off the supernatant and resus. Suspend the beads in 200 microliters of polarizing medium centrifuge the rested cells at 300 G for five minutes at room temperature following the spin take off the super datin and resuspend cells in 200 microliters of polarizing medium containing antibody coupled beads incubate for 72 hours at 37 degrees Celsius in a 5%carbon dioxide incubator without changing medium. The protocol for staining and fixation is described in brief here.
First, the cells are washed in PBS and incubated with fixable dead cell staining solution. After another wash step, the cells are fixed in para formaldehyde following which the cells can be treated without adenovirus biosafety precautions after fixation, the cells are washed again and permeable with methanol. After another wash step, the cells incubated with anti FCR three to block non-specific binding.
Finally, the cells are incubated with anti FCR three and PE coupled anti fox P three antibodies. There is the risk of losing a lot of cells during the fixation and staining procedure due to the multiple washing steps to avoid loss. Make sure to take off the supinate right after centrifugation without disturbing the palette with a piper tip After the cells have been stained, wash the cells twice in PBS and analyze them on a flow cytometer.
A 96 well reader on the flow cytometer allows performing T-cell infection, differentiation, fixation, and staining in the same 96 well plate providing an efficient workflow for parallel analyses of multiple conditions, but the analysis can also be performed in standard fax tubes to assess the effect of adenovirus infection on T-cell viability and treg differentiation. Naive T cells purified from DO 1110 transgenic car delta one transgenic mice were infected with control adenovirus at different MOIs and activated in treg polarizing conditions. Fixed and stained cells were analyzed by flow cytometry for the expression of the infection marker.
GFP incorporation of dead cell die and for the differentiation marker. FOX P three as shown here. Increasing the MOI from one to 50 results in a higher percentage of infected cells.
While neither viability nor treg differentiation is affected to determine whether adenovirus infection affects the activation status of T cells. Infected and non-infected cells were analyzed for expression of activation and resting markers by facts as seen here. Expression of these markers was indistinguishable in infected and non-infected cells when cells were then activated in treg polarizing conditions.
Upregulation of activation markers and downregulation of naive T-cell markers was very similar in infected and non-infected cells taken together. These data indicate that an infection of T cells at a naive state does not interfere with subsequent T-cell activation to test the level of overexpression achieved with the described adenoviral transduction protocol, naive T cells were infected with microRNA 1 55 encoding or control adenovirus at an MOI of 50 cells were either rested for 40 hours, activated for 40 hours, or were rested for 40 hours, followed by an activation of 40 hours. CDNA was generated and micro RNA 1 55 expression was determined relative to snow RNA 202.
By quantitative PCR microRNA 1 55 overexpression levels in naive T cells rested for 40 hours matches endogenous microRNA. 1 55 expression levels. After 40 hours of T-cell activation, activation of those cells still induces activation dependent microRNA 1 55 upregulation.
This demonstrates the high level of overexpression in naive T cells by adenovirus on top of the endogenous activation induced micro RNA expression. Once mastered the procedure of cloning virus generation and T cell infection can be completed within four weeks. The virus, once it's generated, can be stored long term or re amplified within three days.
While attempting this procedure, it is important to remember to carefully control all experiments with an empty vector and to first establish the effect of adenoviral infection itself on the studied process. Please keep in mind that adenoviral infection and overexpression is transient and lasts for only about five to seven days.
