Electroporation of the Jurkat T cell line.

<p>(<b>A</b>) Jurkat cells were electroporated with pT2-GFP in the presence of each of the 7 different buffers. Viability and GFP expression were evaluated by flow cytometry after 24 h. Cell viability is expressed as % of the control mock electroporated condition (100%). Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Statistical analysis was performed using One Way ANOVA and Tukey post test (* = P<0.05). (<b>B</b>) Jurkat cells were electroporated with buffer 3P. Cell viability and GFP expression were observed until day 20. Values in this figure are the average of two separate experiments in triplicate and are expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.05 (*);p<0.01 (**).</p

Electroporation of transposon and transposase maintains transgene expression after cell viability recovery.

<p>PBMCs from two healthy donors were electroporated using 1SM buffer, 20 µg of pT2-GFP plasmid and 2 µg of SB100x transposase. Cell viability and GFP expression were analyzed by flow cytometry on day 1 and 9. Values are the average of two donors in triplicate and are expressed as mean±SEM.</p

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

<div><p></p><p>Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.</p> <p>Aims</p><p>We herein report a convenient and affordable method based on <i>in house</i> made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.</p> <p>Results</p><p>This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as <i>in vitro</i> expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.</p> <p>Conclusions</p><p>We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.</p> </div

Electroporation of resting vs activated mouse primary T lymphocytes.

<p>Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and either directly electroporated or activated for 24 h with anti-CD3/anti-CD28 and then electroporated. Buffer 2S and 4 µg of pT2-GFP plasmid were used. Cell viability and GFP expression were analyzed after 24 h by flow cytometry. Cell viability is normalized to control (not electroporated) cells. Data are representative of two experiments in triplicate and expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.0001 (**) or p = 0.002 (*).</p

Efficiency of murine T lymphocyte electroporation is dependent on different buffer and mouse strain.

<p>(<b>A</b>) Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and electroporated using in house buffers and 4 µg of pT2-GFP plasmid. Cell viability and GFP expression were analyzed after 24 h by flow cytometry. Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Statistical analysis was performed using One Way ANOVA and Tukey post test. P<0.05 (*). (<b>B</b>) Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and electroporated using 2S buffer or Lonza buffer and 4 µg of pT2-GFP. Analysis was performed 24 h later by flow cytometry. Data were analyzed by unpaired Student t test p<0,05 (*). (<b>C</b>) Total lymphocytes from lymph nodes of C57BL/6, Balb/c and B10A mice were isolated and electroporated using 2S buffer and 4 µg of pT2-GFP plasmid. Cell viability was normalized to control (not electroporated) cells. All values in this figure represent the average of two experiments in triplicate and are expressed as mean±SEM.</p

Electroporation efficiency of different buffers in primary human T lymphocytes.

<p>(<b>A</b>) PBMCs from two healthy donors were electroporated using in house buffers and 4 µg of pT2-GFP plasmid. Cell viability and GFP expression were analyzed after 24h by flow cytometry. Cell viability is normalized to control (not electroporated) cells. Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Values are the average of two donors in triplicate and are expressed as mean±SEM. Statistical analysis was performed using One Way ANOVA and Tukey post test (* = P<0.05). (<b>B</b>) Representative dot plots of GFP (pT2-GFP; 4 µg) expression 24 h after electroporation with 1SM buffer. Numbers in plots represents the percentage of cells in the gate. Gray line = negative control; black line = cells electroporated with pT2-GFP. (<b>C</b>) Electroplated lymphocytes were stained for CD4 and CD8 24 h after electroporation with 1SM buffer and 4 µg pT2-GFP plasmid. Data are representative of two donors. Numbers in plots represents the percentage of cells in the gate.</p

T cell electroporation with Chimeric Antigen Receptor (CAR) results in stable gene expression conferring target-specific cytotoxicity.

<p>PBMCs from one donor were electroporated using 1SM buffer, 20 µg of pT3-20z plasmid and 0,5 µg of SB100x transposase plasmid. One day later, lymphocytes were stimulated with irradiated L388 cells and CAR expression was evaluated until d+30 by flow cytometry analysis using an anti-fab antibody. (A) Representative histograms of 20z CAR expression at d+1, d+10, d+20 and d+30 after electroporation (gray line = non electroporated cells stained anti-Fab; black line = lymphocytes electroporated with pT3-20z). (B) Analysis of 20z CAR expression until d+30 summarizing data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#pone-0060298-g008" target="_blank">Figure 8A</a>. (C) Kinetics of expansion of control mock electroporated (Neg) or 20z+ lymphocytes after stimulation with L388 cells. (D) Expanded cells were used in different effector/target (E/T) ratios in a cytotoxic assay against the CD19+/CD20+ Nalm-6 GFP+target cell line; Neg = CTLs not electroporated.</p

Long term expression of GFP after electroporation with buffer 1SM.

<p>PBMCs from two healthy donors were electroporated using 1SM buffer and 4 µg of pT2-GFP plasmid. After 24 h cells were activated with anti-CD3/anti-CD28 and GFP expression was evaluated for 7 days by flow cytometry.</p

MOESM1 of Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives

Additional file 1: Table S1. Current clinical trials using lentiviral systems available on the Journal of Gene Medicine database ( http://www.abedia.com/wiley/vectors.php )

Chromosomal instability increases after induction of neural phenotype by RA in EC cells.

<p>(A) The rate of chromosomal instability presented a 2-fold increase. The bars indicate mean ± S.E.M. of two independent assays, *p<0.05. (B) Analysis of relative DNA content demonstrated that neural cells (RA) presented less DNA than cells incubated with vehicle. RA, <i>all-trans</i> retinoic acid.</p