Name: piggyBac Transposon System Modification of Primary Human T Cells (Video) | JoVE Journal
Uploaded: 2012-11-05T12:00:00.000+00:00
Duration: 10 min 2 s
Description: Learn to stably modify primary human T cells using the non-viral piggyBac transposon system. This method enables long-term transgene expression for immunology research and therapeutic development.

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5 minutes

SS is supported in part by the HHMI Med into Grad Training Grant through the TBMM Program. MHW is supported in part by a career development award from the Department of Veterans Affairs and the generous support of Dr. and Mrs. Harold M. Selzman. This work was also supported in part by NIH lymphoma SPORE grant P50CA126752 and NIH R01 DK093660.

Day 0
1. Isolation of PBMCs from Human Blood
<ol>
	<li>Collect 20 ml of fresh human blood using venipuncture into Na-heparin vacutainer tubes.</li>
	<li>Mix blood and Advanced RPMI 1,640 in 1:1 (v/v) ratio.</li>
	<li>Add 20 ml lymphoprep medium to a 50 ml centrifuge tube (25 &deg;C). Slowly layer 25-30 ml of blood-RPMI 1,640 mix on top of the lymphoprep.</li>
	<li>Centrifuge at 400 x g for 40 min without brakes.</li>
	<li>Collect both distinct and fuzzy layers using a disposable pipette into 10 ml of 1x PBS (25 &deg;C) and bring the volume up to 50 ml with 1x PBS.</li>
	<li>Centrifuge at 450 x g for 10 min.</li>
	<li>Aspirate the supernatant completely and add 20 ml of Advanced RPMI 1,640.</li>
	<li>Centrifuge at 400 x g for 5 min.</li>
	<li>Aspirate the supernatant. Add 10 ml of complete T cell media supplemented with 5 ng/ml rhIL-15 prewarmed at 37 &deg;C.</li>
	<li>Count the number of cells and plate in a 24 well tissue culture coated plate at 2 x 106 cells/well in complete T cell media supplemented with 5 ng/ml rhIL-15 Add sterile water to surrounding wells. Incubate overnight in a humidified incubator at 37 &deg;C, 5% CO2.</li>
</ol>
Day 1
2. Coating Plates with anti-CD28 and anti-CD3 Antibody for Stimulating T cells
<ol>
	<li>Dilute anti-CD28 and anti-CD3 antibodies in sterile water at a concentration of 1 &mu;g/ml each.</li>
	<li>Add 500 &mu;l each antibody solution to 5 marked wells of a non-coated tissue culture 24 well plate.</li>
	<li>Add sterile water to rest of the wells. Wrap the plate in shrink wrap and place in a 4 &deg;C refrigerator.</li>
</ol>
3. Nucleofection of Unstimulated T Cells
<ol>
	<li>Prewarm T cell media at 37 &deg;C and supplement with 5 ng/ml of rhIL-15. Prepare complete nucleofector solution by adding 500 &mu;l of Nucleofector Supplement 1 to 2.25 ml of Nucleofector solution.</li>
	<li>Aliquot 5 &mu;g each of transposon (Zeo-pT-CMV-eGFP) and transposase (pCMV-piggyBac) in a 1.5 ml microfuge tube. Note: it may be necessary to optimize the transposase and transposon DNA amount for optimal gene delivery and to minimize cellular toxicity. The plasmids can be obtained from the authors by request.</li>
	<li>Harvest PBMCs from the 24 well tissue culture plate into a 50 ml tube. Count the number of cells. Save 2 x 106 cells for use as control during flow cytometry.</li>
	<li>Add 7-10 x 106 cells to a 15 ml tube and centrifuge at 400 x g, 5 min, aspirate supernatant and finger-flick the pellet.</li>
	<li>Add 100 &mu;l of T cell complete nucleofection solution to loosened cell pellet.</li>
	<li>Add the solution-cell mixture to the tube containing the plasmids.</li>
	<li>Add the solution-cell-plasmid mixture to the bottom of the nucleofection cuvette. Be careful not to introduce any bubbles.</li>
	<li>Nucleofect the cells using program U-014 (Unstimulated T cells, human, <a href="http://bio.lonza.com/resources/product-instructions/protocols/" target="_blank">http://bio.lonza.com/resources/product-instructions/protocols/</a>).</li>
	<li>Immediately add 500 &mu;l of prewarmed media with rhIL-15 to the cuvette. Transfer the cells to a well of a 24 well plate with 1.5 ml of prewarmed media with rhIL15.</li>
	<li>Incubate overnight in a humidified incubator at 37 &deg;C, 5% CO2.</li>
</ol>
Day 2
4. Nonspecific Stimulation of T Cells
<ol>
	<li>Harvest cells and determine cell numbers. Set aside 0.5 x 106 cells for flow cytometry to determine the frequency of GFP-positive cells. Use the non-transfected PBMCs as controls.</li>
	<li>Aspirate the CD3/28 antibody solution from the non tissue culture coated plate and rinse each well with T cell media.</li>
	<li>Resuspend the nucleofected cells at 0.5 x 106 cells per ml in complete CTL media supplemented with 5 ng/ml rhIL-15. Add 2.0 ml each of the nucleofected cells in 4 wells of the non tissue culture plate. Add 0.5 x 106 non nucleofected cells to the 5th well.</li>
	<li>Incubate for 3 days in a humidified incubator at 37 &deg;C, 5% CO2.</li>
</ol>
Day 5
<ol>
	<li>Harvest the stimulated T cells from the non-coated tissue culture plate.</li>
	<li>Count and replate the cells in a 24 well coated tissue culture plate at 0.7 x 106 cells/ml in T cell media with 5 ng/ml IL-15.</li>
</ol>
Day 7
7. Analysis of Gene Expression
<ol>
	<li>Harvest cells and stain with anti-human CD8 antibody (could also use anti-CD3 and anti-CD4) and analyze with flow cytometry for %GFP expression.</li>
</ol>
Day 8 (Optional)
8. Expansion of T cells
<ol>
	<li>On day 8 after transfection, T cells can be replated in T-cell media with IL-15 at a density of 0.7 x 106 cells per well for further expansion16.</li>
</ol>

<ol><li>Cary, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposon+mutagenesis+of+baculoviruses%3A+analysis+of+Trichoplusia+ni+transposon+IFP2+insertions+within+the+FP-locus+of+nuclear+polyhedrosis+viruses%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.</a> Virology. 172 (1), 156(1989).</li>
<li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assay+for+movement+of+Lepidopteran+transposon+IFP2+in+insect+cells+using+a+baculovirus+genome+as+a+target+DNA%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA.</a> Virology. 211 (2), 397(1995).</li>
<li>Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+transposition+of+the+piggyBac+%28PB%29+transposon+in+mammalian+cells+and+mice%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.</a> Cell. 122 (3), 473(2005).</li>
<li>Saridey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PiggyBac+transposon-based+inducible+gene+expression+in+vivo+after+somatic+cell+gene+transfer%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer.</a> Mol. Ther. 17 (12), 2115(2009).</li>
<li>Nakanishi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((piggyBac+transposon-mediated+long-term+gene+expression+in+mice%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">piggyBac transposon-mediated long-term gene expression in mice.</a> Mol. Ther. 18 (4), 707(2010).</li>
<li>Wilson, M. H., Coates, C. J., George, A. L. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PiggyBac+Transposon-mediated+Gene+Transfer+in+Human+Cells%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">PiggyBac Transposon-mediated Gene Transfer in Human Cells.</a> Mol. Ther. 15 (1), 139(2007).</li>
<li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+PiggyBac+transposon+system+for+the+sustained+genetic+modification+of+human+T+lymphocytes%5BTitle%5D)+AND+%22J.+Immunother%22%5BJournal%5D)">Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes.</a> J. Immunother. 32 (8), 826(2009).</li>
<li>Raja Manuri, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((piggyBac+transposon%2Ftransposase+system+to+generate+CD19-specific+T+cells+for+treatment+of+B-lineage+malignancies%5BTitle%5D)+AND+%22Hum.+Gene+Ther%22%5BJournal%5D)">piggyBac transposon/transposase system to generate CD19-specific T cells for treatment of B-lineage malignancies.</a> Hum. Gene Ther. 21 (4), 427(2010).</li>
<li>Doherty, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperactive+piggyBac+gene+transfer+in+human+cells+and+in+vivo%5BTitle%5D)+AND+%22Hum.+Gene+Ther%22%5BJournal%5D)">Hyperactive piggyBac gene transfer in human cells and in vivo.</a> Hum. Gene Ther. , In Press (2011).</li>
<li>Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hyperactive+piggyBac+transposase+for+mammalian+applications%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A hyperactive piggyBac transposase for mammalian applications.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (4), 1531(2011).</li>
<li>Bonini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+modification+of+T+cells%5BTitle%5D)+AND+%22Biol.+Blood+Marrow+Transplant%22%5BJournal%5D)">Genetic modification of T cells.</a> Biol. Blood Marrow Transplant. 17, Suppl 1. S15-S20 (2011).</li>
<li>Hackett, P. B., Largaespada, D. A., Cooper, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transposon+and+transposase+system+for+human+application%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">A transposon and transposase system for human application.</a> Mol. Ther. 18 (4), 1531(2010).</li>
<li>Galvan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+mapping+of+PiggyBac+transposon+integrations+in+primary+human+T+cells%5BTitle%5D)+AND+%22J.+Immunother%22%5BJournal%5D)">Genome-wide mapping of PiggyBac transposon integrations in primary human T cells.</a> J. Immunother. 32 (8), 837(2009).</li>
<li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PiggyBac-Mediated+Cancer+Immunotherapy+Using+EBV-Specific+Cytotoxic+T-Cells+Expressing+HER2-Specific+Chimeric+Antigen+Receptor%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">PiggyBac-Mediated Cancer Immunotherapy Using EBV-Specific Cytotoxic T-Cells Expressing HER2-Specific Chimeric Antigen Receptor.</a> Mol. Ther. 19 (12), 2133(2011).</li>
<li>Huye, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combining+mTor+Inhibitors+With+Rapamycin-resistant+T+Cells%3A+A+Two-pronged+Approach+to+Tumor+Elimination%5BTitle%5D)+AND+%22Mol.+Ther%22%5BJournal%5D)">Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination.</a> Mol. Ther. 19 (12), 2239(2011).</li>
<li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+%28G-Rex%29%5BTitle%5D)+AND+%22J.+Immunother%22%5BJournal%5D)">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).</a> J. Immunother. 33 (3), 305(2010).</li>
<li>Kahlig, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+transposon-mediated+stable+gene+transfer+in+human+cells%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Multiplexed transposon-mediated stable gene transfer in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (4), 1343(2010).</li>
</ol>

No conflicts of interest declared.

The method described herein enables stable transgene modification of primary human T lymphocytes. We have previously tested the use of the piggyBac transposon system to modify T cells to express a reporter gene (for more than 4 weeks), a non-immunogenic suicide gene, a chimeric antigen receptor for adoptive immunotherapy (for more than 100 days), and to engineer resistance to immunosuppressive medications7,13-15. Non-viral modification of T cells for adoptive immunotherapy and other applications shoul...

<table><tbody><tr><td>Lympholyte</td><td>Cedarlane</td><td>CL5015</td><td></td></tr><tr><td>Advanced RPMI 1,640</td><td>LifeTechnologies</td><td>12633020</td><td></td></tr><tr><td>Hyclone Fetal Bovine Serum</td><td>Fisher Scientific</td><td>SH3008803</td><td></td></tr><tr><td>GlutaMAX-I Supplement</td><td>LifeTechnologies</td><td>35050-061</td><td></td></tr><tr><td>Human IL-15 Recombinant Protein</td><td>eBioscience</td><td>14-8159</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Amaxa Nucleofector</td><td>Lonza</td><td>AAD-1001S</td><td></td></tr><tr><td>Human T Cell Nucleofector Kit</td><td>Lonza</td><td>VPA-1002</td><td></td></tr><tr><td>CD8-APC</td><td>Southern Biotech</td><td>9536-11</td><td></td></tr><tr><td>Anti-Human CD3</td><td>eBioscience</td><td>16-0037-81</td><td></td></tr><tr><td>Anti-Human CD28</td><td>BD Pharmingen</td><td>555725</td><td></td></tr><tr><td>24 Well Tissue Culture Treated Plate</td><td>BD Falcon</td><td>353047</td><td></td></tr><tr><td>24 Well Non Tissue Culture Treated Plate</td><td>BD Falcon</td><td>351147</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Complete T cell media composition&lt;/strong&gt;&lt;br/&gt; 1x Advanced RPMI 1,640&lt;br/&gt; 5% Heat Inactivated Fetal Bovine Serum&lt;br/&gt; 2 mM GlutamaxIM-I</td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

A schematic demonstrating the steps in genetically modifying human T lymphocytes with a reporter gene (eGFP) is shown in Figure 1. These plasmids are available upon request from the authors. A schemtic demonstrating the steps in genetically modified human T lymphocytes with a reporter gene (eGFP) is showin in Figure 2. It is necessary to activate T cells in order to get them to divide, expand, and propagate in culture. Modified human T cells were then cultured and analyzed using flow cyt...

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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.

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