23, 13801390.10.1038/mt.2015.71 Set up the RNP formation reaction as follows. Competitor B electroporation: 25 mV, 96 F. Transgene expression can be enriched by using G418 and is retained after in vivo growth. Acad. After G418 selection and withdrawal, GFP expression remained stable in NIH3T3 cells for 15 days (Figure S15 in Supplementary Material). Please login or create an account. doi:10.1158/1535-7163.MCT-07-0009, Bonamino, M., Serafini, M., DAmico, G., Gaipa, G., Todisco, E., Bernasconi, S., et al. As shown in Figure 5A, the best electroporation score for MSC was obtained using buffer 2S, with 57% of viable cells and 39% of GFP expression. Cells were plated to expand MSCs at 3 104 cells/cm2 density with low-glucose Dulbeccos modified Eagles medium (DMEM Low-glucose, Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) and 100 U/ml penicillin and 100 g/mL streptomycin (Sigma-Aldrich, MO, USA). Co-electroporation of 293T cells with the report construct and the plasmid carrying CRISPR/Cas9/gRNA, but not CRISPR/Cas9 lacking the gRNA sequence, resulted in GFP expression in approximately 7% of the RFP+ cells (3% out of 42%), indicating that sequence-specific DNA editing was achieved (Figure 7A). The indels are represented by lower case characters; numbers inside parenthesis depict substitutions (~) and numbers outside parenthesis depict additions (+) or deletions (). Furthermore, our results are comparable to those reported in the literature for cell lines like K562 (Gresch et al., 2004) and primary MSCs (Aluigi et al., 2006), although direct comparison of the results must be taken carefully because different plasmids were used. It allows for highly efficient transfer of various substrates directly into the nucleus of cells. It enables highly efficient, transfection of primary cells, stem cells, neurons, and cell linesthat have traditionally been difficult to transfect via electroporation and other non-viral transfection methods. Cell Biol. Sleeping beauty-based GFP gene transfer to adipose tissue derived human mesenchymal stem cells (MSCs). Electroporation is a nonviral method for gene transfer that is demonstrating encouraging results, being successfully used for the manufacture of antitumor lymphocytes ( Ramanayake et al., 2015) and other applications ( Kotnik et al., 2015 ), but the mechanism of DNA/RNA transfer is not fully understood ( Satkauskas et al., 2012 ). doi:10.1146/annurev-bioeng-071813-104622, Yin, H., Kanasty, R. L., Eltoukhy, A. Only registered User can view this video. doi:10.3109/08830185.2014.917412, Chicaybam, L., Sodre, A. L., Curzio, B. Electroporation strategies using Chicabuffers were recently successfully applied to colon cancer cell lines (de Souza et al., 2013) and human mesenchymal stem cells (MSC; unpublished data). Safety and tumor responses with lambrolizumab (AntiPD-1) in melanoma. (2013). Clinical Scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. Vadeikien R, Jaktys B, Ugenskien R, atkauskas S, Juozaityt E. Biomedicines.
Frontiers | An Efficient Electroporation Protocol for the Genetic The use of electroporation for the genetic modification of cells is being adopted by many laboratories as it represents a fast and cheap option for transfer of plasmids and RNA. . Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model. Electroporation of CRISPR/Cas9 cassettes promotes gene editing of PBMCs and 293T cells. doi:10.1038/nmeth846, Vargas, J. E., Salton, G., Sodr de Castro Laino, A., Pires, T. D., Bonamino, M., Lenz, G., et al. LC, CB, BP, MC, PR, and LB performed the electroporation experiments (cell lines, MSCs, and PBMCs) and data analysis and interpretation. When using SB100, long-term expression of GFP using this buffer was seen in 12% of cells (Figure 5B). 0000006312 00000 n
Viability and GFP expression were followed for 10 days (suspension cell lines) or 7 days (adherent cell lines), with some cells retaining high levels of GFP (K562, HEL, B16F10) and others showing low expression of the marker after the expansion (NIH3T3, Jurkat, P815) (Figures S114 in Supplementary Material). (C) 5 105 B16F10 cells submitted or not to selection with G418 were injected in the left flank of C57Bl/6 mice. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. Nat. A., Kim, S., et al. doi:10.1056/NEJMoa1305133, Hausl, M. A., Zhang, W., Mther, N., Rauschhuber, C., Franck, H. G., Merricks, E. P., et al. The use of PBMCs from healthy donors was approved by an IRB (Brazilian National Cancer InstituteINCAEthics Committee), and donors signed review board approved informed consents. All the authors read and approved the final manuscript. Methods 3, 109116. Hold at 4C. Viability (blue bar), GFP expression (green bar), and electroporation score (red bar) were assessed 1 day after nucleofection (d + 1). G418 was added 2 days after nucleofection, and GFP expression was accompanied until d + 20 (NIH3T3) or d + 12 (B16F10). Acad. The volumes below will allow for one (1) electroporation (with the addition of cells) of 25 l. Unauthorized use of these marks is strictly prohibited. HEK293FT and PBMCs were electroporated with pX330-PDCD-1 (10 g) and pRGS-CR-target (5 g). 2011 Sep;22(9):1043-51. doi: 10.1089/hum.2011.143. The gRNA used for PDCD1 locus edition in our report targets exon 2, in contrast to exon 1 editions promoted by Schumann et al. and transmitted securely. -. Place SOC recovery medium in a 37C water bath. Epicentre QuickExtract DNA Extraction Solution (Epicentre #QE09050), 65C for 15 min Electroporation is a physical transfection method that permeabilizes the cell membrane by applying an electrical pulse and moves molecules via the electrical field into the cell. doi:10.1517/14712598.2012.654775, Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., et al. Using a square-wave pulse technology, Lonzas Nucleofector electroporator was shown to be very efficient in several cell lines and primary human and murine cells, inducing high expression of the transgene and substantial viability. These vectors and others recently reported in the literature (Kowarz et al., 2015), in conjunction with Chicabuffers, could be potentially used in diverse experimental gene therapy approaches, such as T cell immunotherapy (Singh et al., 2015), MSCs (Martin et al., 2014), and stem cell gene therapy protocols (Aiuti et al., 2013), further facilitating the application of these technologies in basic, translational, and clinical studies. EnGen Lba Cas12a (Cpf1) (NEB #M0653) DMEM with Glutamax (or appropriate growth medium) with 10% FBS Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. J. Natl. The results of gene editing experiments in 293T and PBMCs are summarized in Figure 7C. The following additional materials are required but not supplied: horizondiscovery.com Electroporation instrument Electroporation reagents (buffer, cuvettes, transfer pipettes) Natl. Add 50 l of Epicentre QuickExtract DNA Extraction Solution and shake/vortex for 5 minutes. 2019 Apr;30(4):511-522. doi: 10.1089/hum.2018.218. Front. Summarized electroporation conditions for each cell line (based in Figure 2; d1 after electroporation). Hum Gene Ther. To achieve efficient gene editing of target cells, Cas9 nuclease and the gRNA must be expressed in the cell, ideally in a transient fashion. )`)(kd)Jom!7v>?Y BM* 0oB^~+VN+Xj8'PY,dURO-'2sJ{RTLuQ %^mB4c
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<. This protocol is written for use with the 16-well Nucleocuvette. These results suggest that Chicabuffers can be used for CRISPR genome editing in different cell lines and primary cells, including large-scale screening of different gRNAs. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Electroporation using square-wave generating devices, like Lonzas Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. Please enable it to take advantage of the complete set of features! Importantly, after 24 h of electroporation, the cells showed a good viability (Figure 2), allowing expansion and recovery from the nucleofection. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. NUCLEOCUVETTE is a trademark of Lonza Group Ltd. Pre-warm selective plates at 37C for 1 hour. In summary, our study describes general guidelines for the efficient electroporation of primary mammalian cells and several cell lines. (2013). This result suggests that no gene silencing occurs for the SB transgenic cassette, supporting in vivo utilization of this tool, as described elsewhere (Belur et al., 2003; Hausl et al., 2010). Efficient nonviral sleeping beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity. doi:10.1371/journal.pone.0047868, Gillet, J.-P., Varma, S., and Gottesman, M. M. (2013). Our results show the feasibility of this approach, enabling a stable transgene expression in CD34+ cells from cord blood samples, keeping GFP expression throughout hematopoietic differentiation. 0000003101 00000 n
Nature 483, 603607. Desired culture plate, DNA Extraction and Genome Editing Analysis, EnGen Mutation Detection Kit (NEB #E3321) South China University of Technology, China, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, China, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (CAS), China, University of California, Davis, United States. pLR: a lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters. Home Protocols Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP) into adherent cells using the Lonza 4D-Nucleofector Overview: EnGen Lba Cas12a (Cpf1) (NEB # M0653) is a nuclease that may be used in vivo to create targeted genome modifications. FACSCalibur (BD Bioscience) was used to perform morphologic evaluation of viability (FSC vs. SSC) and GFP expression analysis. After 3 days, the cells were recovered and analyzed by flow cytometry for GFP expression. A., and Bonamino, M. H. (2013). For the creation of pT3-Neo-EF1a-GFP plasmid, GFP was excised from pT3-GFP by digestion with AgeI/NotI, and the neomycin resistance gene (NEO), which was synthesized by Genscript (Piscataway, NJ, USA), was inserted.
Optimization of the transfection of human THP-1 macrophages by - PubMed National Library of Medicine 0000022740 00000 n
The pT2-GFP and SB100X (Mts et al., 2009) constructs were kindly provided by Dr.
An Efficient Electroporation Protocol for the Genetic - PubMed Are you interested to see our Nucleofector Devices live in action and how they perform? PLoS ONE 8:e74994.
Easy and robust electrotransfection protocol for efficient - Nature 33, 402416. Learn more by visiting our 4D-Nucleofector X Unit product page or by watching the video. Federal government websites often end in .gov or .mil. Section 1 Background. The long-term levels of GFP expression did not correlate with GFP expression at early days after nucleofection, suggesting that the cell lines have different intrinsic susceptibilities to SB-induced transgene integration. These cells were plated in methylcellulose-based medium, allowing long-term assessment of GFP expression and differentiation potential. 2006 Oct;34(10):1333-43. doi: 10.1016/j.exphem.2006.05.023. Would you like email updates of new search results? A., Kim S., et al. doi:10.1021/ar200213g, Belur, L. R., Frandsen, J. L., Dupuy, A. J., Ingbar, D. H., Largaespada, D. A., Hackett, P. B., et al. One of the areas that benefited the most with the use of cell lines was cancer research, with the derivation of several cell lines that can be used as models for different cancers. This setting could be used to co-electroporate a plasmid encoding a reporter gene (or Cas9 nuclease) and multiple short RNAs (such as gRNAs for editing several loci). Download more detailed guideline on Genome Editing using Nucleofector Technology. TheraPEAK Nucleofector Products for the 4D-Nucleofector LV Unit help you expedite translating your non-viral gene modification step into a GMP process. Viruses. (A) MSCs were electroporated with each one of the seven buffers and the recommended program. High transfection efficiencies of up to 90% for plasmid DNA and 99% for oligonucleotides, like siRNA. Cancer Ther. Springer International Publishing, 717757. Seed the cells so that they will be around 70-90% confluent on the day of transfection. 15, 541555. Please refer to the Lonza 4D-Nucleofector manual for proper usage of the equipment. 33, 480488. The recent description of the CRISPR/Cas9 system as an efficient tool to edit the genome of cells has clear implications for basic cell biology studies and gene therapy protocols (Doudna and Charpentier, 2014). Electroporation score for cell lines. Cells were transferred to a sterile 0.2-cm cuvette and electroporated using the reported program (Table 1) of Lonza Nucleofector II electroporation system. (2012). (A) 293T cells were electroporated (buffer 3P, program A-023) without plasmid (negative control), with pRGS-CR plasmid (without PDCD1 target sequence; mock), or with pRGS-CR-PDCD1. Careers. 0000008082 00000 n
Here we present a method for the introduction of Cas12a RNPs into HEK293 FT cells using the Lonza 4D-Nucleofector Electroporation System. Sci. Science 337, 816821. Science 346, 1258096. doi:10.1126/science.1258096, Faget, D. V., Lucena, P. I., Robbs, B. K., and Viola, J. P. B. Sleeping beauty-based GFP gene transfer to human cord blood CD34+ cells. 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. Science 341, 1233151.10.1126/science.1233151 Genome editing. As negative controls, we electroporated cells only with pT3-NEO-EF1a-GFP. Protocol. Epub 2013 Mar 26. doi:10.1038/mt.2010.47, Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., et al. See this image and copyright information in PMC. This method uses a guide RNA to protein ratio of 10:1. Two experiments were done for each cell. Figure 4. We have not found it necessary to account for volume overage. These cells are used to model disease in vitro and in vivo, providing information about oncogenesis-related pathways and insights into therapeutic strategies (Gillet et al., 2013). doi:10.1073/pnas.2331323100, Park, E. S., Rabinovsky, R., Carey, M., Hennessy, B. T., Agarwal, R., Liu, W., et al. After 15 days, we excised the tumor and plated the cells in 25 cm2 culture flasks. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. (2006). N. Engl. It is a powerful tool for transfecting large DNA fragments and achieving good transfection efficiencies in cell lines. doi:10.1038/nrg3686, Kim, H., Um, E., Cho, S.-R., Jung, C., Kim, H., and Kim, J.-S. (2011). 0000004777 00000 n
Add 10.5 l of cells to each 14.5 l RNP reaction. To assess viability of adherent cell lines, cells were plated in triplicate in 96-well microtiter plates immediately after electroporation. You have been idle for more than 20 minutes, for your security you have been logged out. doi:10.1038/gt.2014.26, Bilal, M. Y., Vacaflores, A., and Houtman, J. C. (2015). Natl. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Bookshelf Ther. doi:10.1038/nature11003, Beane, J. D., Lee, G., Zheng, Z., Mendel, M., Abate-Daga, D., Bharathan, M., et al. bioRxiv. Data from electroporation experiments were analyzed by one-way ANOVA followed by Tukeys multiple comparison test using GraphPad Prism 6 software. Clipboard, Search History, and several other advanced features are temporarily unavailable. 0000014045 00000 n
The cell suspension was centrifuged at 400 g, room temperature, for 10 min, and the pellet was resuspended on PBS, followed by filtration with 100-m mesh strainers. The study was approved by the local Research Ethics Committees. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). (2015). Multiple target editing is possible using CRISPR systems. The plate was read on a spectrophotometer at 595 nm (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). However, due to high toxicity traditional electroporation has been less successful for efficiently transfecting more biologically relevant primary cells and stem cells, which has limited its application. (2012). Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses. With its superior transfection performance, Nucleofection offers various advantages over traditional electroporation methods: Electroporation usually requires cells to be in suspension for transfection. No use, distribution or reproduction is permitted which does not comply with these terms. Synthetic gene transfer vectors II: back to the future. Adherent cell lines showed GFP values slightly lower (3065%), with Hela showing the best result with 66.4 8.3% of GFP expression using buffer 3P. Learn about CRISPR from discovery to cell and gene therapies, covering topics such as finding therapeutic pathways or process development considerations for bringing gene edited therapies to the clinic. Molecular evolution of a novel hyperactive sleeping beauty transposase enables robust stable gene transfer in vertebrates. (2004). The electroporation score was calculated based on cell viability (after normalization against the viability of non-transfected cells) and transgene expression on d + 1, and the score set to the formula Viability (%)*Expression (%)/F. A division factor (F = 50 for adherent cell lines and F = 100 for non-adherent cell lines) was used in the score formula to fit the results in the graph scale. Liver Cells for ADME & Toxicology Studies, Endotoxin Detection and Analysis Software, MODA-ES The Next Generation Electronic Batch, MES for Cell and Gene Therapy Manufacturing, higher transfection efficiency than other non-viral transfection methods, Maasho et al 2004 Journal of Immunological Methods, Marques and Williams, 2005 Nature Biotechnology, Optimized protocols offering comprehensive guidance for optimal Nucleofection. Acc.
Electroporation-based applications in biotechnology. In addition, we show that the level of transfection achieved using Chicabuffers allows efficient genomic edition of the potentially clinical relevant PD1 locus in human cells, such as 293T and PBMCs, using the recently described CRISPR/Cas9 system (Jinek et al., 2012). Sang Wang Han (UNIFESP, Brazil). doi:10.1371/journal.pone.0074994, Doudna, J. The HT Nucleofector System is an independent platform offering high-throughput transfection in 384-well format. Rev. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Electroporation-based technologies for medicine: principles, applications, and challenges. Add 75 l of media to the cells in the cuvette, pipetting gently. The other cell lines showed only a modest increase in GFP-positive cells at day 30, ranging from 2% (BA/F-3) to 12% (K562). Trypsin to release cells We also offer solutions for automation, site-directed mutagenesis, as well as your favorite restriction enzyme, ligase or competent cell products. NFAT1 C-terminal domains are necessary but not sufficient for inducing cell death. Nonviral gene delivery with the sleeping beauty transposon system. 0000007521 00000 n
Transfection efficiency was monitored by flow cytometry after 24 hours. (2008). J. Genet. MeSH The GFP expression can be restored by CRISPR-mediated non-homologous end joining (NHEJ) repair.
An Efficient Low Cost Method for Gene Transfer to T Lymphocytes Figure 7. government site. Mol. Generic cuvettes were used for all the electroporations (Mirus Biotech, Madison, WI, USA cat. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), Fundao de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES), Brazilian National Cancer Institute (INCA), and Oncobiology program/Universidade Federal do Rio de Janeiro (UFRJ). This results place Chicabuffers as a valuable tool for cheap and fast gene modification of basically every cell tested, with important potential applications in cell therapy and development and testing of synthetic circuits in mammalian cells. Sleeping beauty-mediated modification of cells as described here proved to be stable in vitro and in vivo, with cells retaining transgene expression during tumor development in immunocompetent mice. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee. Calculation was based on the formula % = 100 [OD for control (non-electroporated) cell line/(OD for control (non-electroporated) cell line + OD for electroporated cell line)]. Furthermore, we showed efficient CRISPR-mediated genome editing of PDCD1 gene in 293T and human PBMCs electroporated using Chicabuffers. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. Resuspend the cells in 5-10 ml of media. Annu. The characterization of indels in PBMCs and 293T cells indicate that the use of our optimized electroporation protocol allowed efficient editing of PDCD1 locus in the tested samples. -, Behr J.-P. (2012).
Electroporation - an overview | ScienceDirect Topics CD34; CRISPR; GFP; MSC; PD-1; T lymphocyte; cell line; electroporation; transposon. As showed in Figure 2, the majority of cell lines showed high electroporation scores independent of the buffer, with exception of P815, which showed an overall low efficiency but demonstrated best performance with buffer 3P. doi:10.1016/j.tibtech.2015.06.002, Kowarz, E., Lscher, D., and Marschalek, R. (2015). Their unlimited proliferative capacity, high degree of homogeneity, and relatively easy maintenance in culture allow the generation of large number of cells required for testing numerous candidate drugs (Barretina et al., 2012), -omics profiling (Nishizuka et al., 2003; Griffin and Shockcor, 2004; Blower et al., 2007), and signaling pathways studies (Park et al., 2010), to cite some examples. Cells were left resting in RPMI + 10%FCS for 24 h at 37C and 5% CO2 and then evaluated by flow cytometry using ACCURI C6 (BD Bioscience). Long-term transgene expression in electroporated cell lines using sleeping beauty system. Our solution is an improved electroporation technology, the Nucleofector Technology, originally introduced into the market by legacy Amaxa in 2001. Representative flow cytometry plots are depicted in Figure 1, showing 7AAD staining and GFP signal (gated in 7AAD negative cells) for a high electroporation score cell line (HEL) and FSC/SSC and GFP signal for a low score cell line (NIH3T3).
Electroporation Protocol (C2986) | NEB We describe here an efficient general protocol for electroporation based modification of T lymphocytes. Curr Gene Ther. The colonies were identified and quantified using STEMvision (Stem Cell Technologies, Inc.) for the burst-forming units-erythroid, colony-forming units-erythroid, colony-forming units-granulocyte or macrophage or granulocyte-macrophage, and colony-forming units-granulocyte/erythroid/megakaryocyte/macrophage. doi:10.1016/j.ymeth.2003.11.011, Kim, H., and Kim, J.-S. (2014). doi:10.1073/pnas.1512503112, Singh, H., Moyes, J. S. E., Huls, M. H., and Cooper, L. J. N. (2015). For CD34+ cells separation, mononuclear cells (MNCs) were isolated from umbilical cord blood after Ficoll density gradient using the same protocol above described. (C) Summarized results obtained for 293T cells and PBMCs, showing the number of colonies sequenced and the percentage of indels detected. a method in which CELLS are subjected to an electrical impulse that leads to the temporary formation of pores in the cell MEMBRANE. Electroporation provides a means of transforming cells (see TRANSFORMATION ). Whether you are performing your first cloning experiment or constructing multi-fragment DNA assemblies, NEB has the solution for you. 8, 501507. Cell lines were electroporated with pT2-GFP (4 g) using the combination of buffer and program indicated on Table 1, with (white bar) or without (black bar) the addition of SB100 transposase (1 g). doi:10.1016/j.jcyt.2015.05.013, Sartore, R. C., Campos, P. B., Trujillo, C. A., Ramalho, B. L., Negraes, P. D., Paulsen, B. S., et al. 12, 275286.
Does anyone have an optimized protocol for electroporation human dermal 369, 134144. Nonviral methods like liposomes and electroporation show varying efficiencies, with several cell lines and primary cells showing poor transfection rates and cell death (Wang et al., 2012; Yin et al., 2014). Gene Ther.
Electroporation Definition & Meaning - Merriam-Webster Data were analyzed using the FlowJo software (Tree Star). Figure 2. doi:10.1007/978-3-319-10320-4_23, Martin, P. K., Stilhano, R. S., Samoto, V. Y., Takiya, C. M., Peres, G. B., da Silva Michelacci, Y. M., et al. (2015), showing that different gRNAs can be used to efficiently disrupt the PDCD1 gene sequence. Some of the tested cells represent classical cellular models for ectopic gene expression (293T, NIH-3T3), cell signaling (Jurkat and 293T), growth factor dependence (BA/F-3), or simply relevant cells in terms of therapy and cell differentiation (MSCs, PBMCs and Cord Blood CD34+ cells). Synthetic gene transfer vectors II: back to the future. Rev. 00-6693) was performed immediately before FACS acquisition following manufacturer instructions. Exp Hematol. (2013). Long-term expression of the transgene can be potentially increased by the use of SB100 RNA, decreasing the toxicity of the electroporation process as reported (Peng et al., 2009), or by carefully titrating the transposase plasmid mass to avoid overproduction inhibition (Grabundzija et al., 2010).