The overall goal of this procedure is to stably gene, modify primary human T cells with a non-viral transposon system. First, collect human blood and then isolate the peripheral blood mononuclear cells proceed to nucle effect the pbmc with the piggyback transpose on system and a transgene of interest like EGFP. Next, stimulate the T cells to proliferate in culture.
Then analyze the T cells for transgene expression. Ultimately, flow cytometry analysis is used to show stable transgene expression in T cells modified with the piggyback transpo on system. Unlike plasma transfection of human T cells, transposons have the advantage of stably integrating transgenes for long-term expression.
The trick is to follow a very specific protocol in handling the P BMCs during isolation and after nucleo affection, we first implemented this method to make cultured human stable cell lines for expressing different genes. This approach helps to answer key questions in the immunology field, such as how one can modify T-cells to make them function better. Demonstrating the procedure will be sunon and saha, a graduate student from my laboratory Using venipuncture.
Collect 20 milliliters of fresh human blood into sodium heparin vacutainer tubes. Mix the blood with advanced RPMI 1640 media in a one-to-one ratio. Then add 20 milliliters of lympho prep medium to a 50 milliliter centrifuge tube.
Slowly layer 25 to 30 milliliters of the diluted blood on top of the lympho prep and centrifuge at 400 times G for 40 minutes without breaks. Transfer both the distinct and the fuzzy layers into 10 milliliters of room temperature. PBS Bring the total volume up to 50 milliliters with PPBS.
Then harvest the cells by centrifugation at 450 times G for 10 minutes, aspirate the supernatant completely, then wash the cells once with 20 milliliters of advanced RPMI after centrifugation Resus. Suspend the cell pellet in 10 milliliters of complete T-cell media. Supplemented with five nanograms per milliliter of recombinant human IL 15 enumerate the cells now in a 24 well tissue culture coated plate seed, two times 10 to the sixth cells per well.
Then add a sterile water to any surrounding wells. Incubate the P BMCs overnight in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. Dilute the anti CD 28 and anti CD three antibodies in sterile water to a concentration of one microgram per milliliter.
Each add 500 microliters of each antibody solution to five marked wells of a non coated 24 well tissue culture plate. Then add sterile water to the rest of the wells. Wrap the plate and place at four degrees Celsius.
Prewarm the T-cell media at 37 degrees Celsius and supplement with five nanograms per milliliter of recombinant human IL 15. Also prepare fresh complete nucleo effector solution. Now aliquot five micrograms each of transposon and transpose A in a 1.5 milliliter fuge tube.
It may be RY to optimize the transpose A and transposon DNA mount for optimal gene delivery. And to minimize cellular toxicity, Harvest the PBM CS from the 24 well tissue culture plate into a 50 milliliter tube. After counting the cells, save two times 10 to the sixth P BMCs for use as a control during flow cytometry.
Next, add between seven and 10 million cells to a 15 milliliter tube and centrifuge at 400 times G for five minutes. Aspirate the supernatant and finger flick the pellet. Add 100 microliters of T-cell complete nucle affection solution to the loosened cell pellet.
Now add the solution cell mixture to the plasmids. Next, add the solution cell plasmid mixture to the bottom of the nucle affection vete. Be careful not to introduce any bubbles Nucle affect the cells using program U dash 0 1 4.
For unstimulated human T cells transfer the cells to a well containing 1.5 milliliters of pre-war media plus R-H-I-I-L 15 incubate the nucle affected unstimulated T cells in a cell culture incubator overnight. After harvesting the cells determine the cell numbers set aside 0.5 times 10 to the sixth of the non transfected pbmc as controls for the flow cytometry determination of the frequency of GFP positive cells. Then aspirate the CD 3 28 antibody solution from the non tissue culture coated plate and rinse each well with T-cell media resuspend.
The nucle affected cells at 0.5 times 10 to the sixth cells per milliliter. In complete CTL media, supplemented with five nanograms per milliliter of RH IL 15 add 2.0 milliliters. Each of the nucle affected cells in four wells of the non tissue culture plate.
Add 0.5 times 10 to the sixth. The nonnuclear affected cells to the fifth. Well incubate the plate for three days in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.
On day five, harvest the stimulated T cells from the non coated tissue culture plate. Then in a 24 well coated tissue culture plate at 0.7 times 10 to the six cells per milliliter in T-cell media containing five nanograms per milliliter of IL 15. After harvesting the cells immuno stain for either CD eight, CD three, or CD four for percent GFP expression, analyze the cell preparations with flow cytometry if expansion of the T cells is required.
Then on day eight, after transfection plate cells at a density of 0.7 times 10 to the sixth cells per well in T-cell media containing IL 15 for piggyback mediated gene modification of human T cells. Primary human cells are transfected with two plasmids. The transposes plasmid drives constitutive expression of piggyback protein under A CMV promoter.
The transpo on plasmid contains the GFP reporter and the piggyback inverted repeats needed for gene transfer. Stable GFP transgene expression in T cells modified with the piggyback transposon system can be studied by fax analysis. Here, cells were stained with a PC conjugated, anti CD eight and analyzed for EGFP and A PC fluorescence on day one.
EGFP expression is detected in 76.4%of the CD eight positive cells. The decrease in EGFP expression between day one and day seven is likely due to the fact that not all transfected cells undergo stable integration of transpose on DNA. You should now have a good understanding of how to isolate pbmc and stably gene.
Modify them with the piggyback transpose on system. Don't forget to use sterile technique when performing phlebotomy. Work quickly to preserve cell viability.
Following this procedure, other methods like a T-cell cytotoxicity assay can be performed in order to determine antigen specificity of the T-cells. Future experiments will explore the use of transposons for T-cell based immunotherapy for cancer and mice.
