Figure 2. Transgene expression can be enriched by using G418 and is retained after in vivo growth. 0000020620 00000 n Proc. doi:10.1371/journal.pone.0047868, Gillet, J.-P., Varma, S., and Gottesman, M. M. (2013). Suspension cell lines showed the best results regarding GFP expression, in which values above 60% were recurrently obtained. Resuspend the cells in 5-10 ml of media. doi:10.1146/annurev-bioeng-071813-104622, Yin, H., Kanasty, R. L., Eltoukhy, A. and transmitted securely. With the objective of determining the best-suited buffer for the electroporation of each cell line, cells were electroporated with seven different buffers and the viability and GFP expression were analyzed. We describe here an efficient general protocol for electroporation based modification of T lymphocytes. doi:10.1038/nrg3686, Kim, H., Um, E., Cho, S.-R., Jung, C., Kim, H., and Kim, J.-S. (2011). Does anyone have an optimized protocol for electroporation human dermal fibroblast with Lonza 4D machine? Sang Wang Han (UNIFESP, Brazil). Due to this independency from cell proliferation, it allows for efficient transfection of even non-dividing primary cells (like resting T cells or neurons) and also speeds up expression. Find out more by visiting the HT Nucleofector System product pageor watching our video tutorial. The use of PBMCs and CD34+ cells from healthy donors was approved by an IRB (Brazilian National Cancer InstituteINCAEthics Committeeprotocol 153/13), and donors signed review board approved informed consents. (2006). Nat. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee. Mol. Add 75 l of media to the cells in the cuvette, pipetting gently. The electroporation protocol for each cell line is summarized in Table 1. Trypsin to release cells 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. 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. The Nucleofector Technology offers a higher transfection efficiency than other non-viral transfection methods (including traditional electroporation1) along with other significant advantages outlined below. (2007). doi:10.1002/biot.201400821, Kuystermans, D., and Al-Rubeai, M. (2015). Cell lines were electroporated with pT2-GFP (4 g) using each one of the seven buffers and the recommended program. Before An efficient low cost method for gene transfer to T lymphocytes. Cells were transferred to a sterile 0.2-cm cuvette and electroporated using the reported program (Table 1) of Lonza Nucleofector II electroporation system. It is a powerful tool for transfecting large DNA fragments and achieving good transfection efficiencies in cell lines. Moreover, the use of electroporation is associated with extensive testing of electric parameters (pulse amplitude, volts) in order to optimize the protocol. Seed the cells so that they will be around 70-90% confluent on the day of transfection. doi:10.1038/nature11003, Beane, J. D., Lee, G., Zheng, Z., Mendel, M., Abate-Daga, D., Bharathan, M., et al. Upon application of an electric field, the cell membrane is compromised,. 0000004503 00000 n After reaching 80% confluence, G418 (Life Technologies) antibiotic was added at 2,000 g/mL. A., and Charpentier, E. (2014). 1121, 323. Transfected samples may be plated in any number of replicates. 18, 18961906. pRGS-CR (Kim et al., 2011) was provided by Dr. Amilcar Tanuri (Federal University of Rio de Janeiro, Brazil), and PDCD1 target sequence cloned in EcoRI/BamHI sites, between a red fluorescent protein (RFP) and a GFP, resulting in an out-of-frame GFP. Data are shown as mean SD from two experiments. Variations of the pT3-Neo-EF1a-GFP construct were developed, such as the pT3-Neo plasmid, which confers resistance to G418 antibiotic and has restriction sites that allow cloning of a second expression cassette. 2022 Dec 2;14(12):2700. doi: 10.3390/pharmaceutics14122700. Exp Hematol. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. 0000003101 00000 n Claudin-3 overexpression increases the malignant potential of colorectal cancer cells: roles of ERK1/2 and PI3K-Akt as modulators of EGFR signaling. Cancer Ther. Ther. Hollis RP, Nightingale SJ, Wang X, Pepper KA, Yu XJ, Barsky L, Crooks GM, Kohn DB. For cells in which the levels of transgene expression was low, we developed sleeping beauty (SB)-based transposon plasmids engineered to confer drug resistance, allowing fast and efficient drug-based selection of cells representing fractions of the cell culture. doi:10.1073/pnas.2331323100, Park, E. S., Rabinovsky, R., Carey, M., Hennessy, B. T., Agarwal, R., Liu, W., et al. A guide to genome engineering with programmable nucleases. Calculate the number of cells you will need for the entire experiment (1-2 x 10. Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon. Desired culture plate, DNA Extraction and Genome Editing Analysis, EnGen Mutation Detection Kit (NEB #E3321) Gene Ther. 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). Genetic modification of cells is a cumbersome and expensive process, often involving the use of viral vectors to achieve high efficiency transgene expression. Sleeping beauty-based GFP gene transfer to human cord blood CD34+ cells. 2022 Oct 24;10(11):2687. doi: 10.3390/biomedicines10112687. Here we present a method for the introduction of Cas12a RNPs into HEK293 FT cells using the Lonza 4D-Nucleofector Electroporation System. Lonzas buffers were already described to have good results when tested with alternative nucleofector IIb programs (Gresch et al., 2004), suggesting that there is still room for optimization of electroporation conditions, reinforcing the potential of testing Chicabuffers under different experimental settings. 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. In recent years, this has opened novel opportunities for disease research and therapeutic development, including the advancement of gene therapies, immunotherapies, and stem cell generation. doi:10.1371/journal.pone.0074994, Doudna, J. A., Kim, S., et al. 0000000876 00000 n Furthermore, when the modified B16F10 cells were injected in vivo and allowed to form subcutaneous tumors, the cells extracted from the tumor at d + 14 post inoculation (dpi) still expressed high levels of GFP, indicating that the transgenic cassette is integrated in the genome and has stable expression, with no signs of in vivo silencing of the transgene (Figure 4C; Figure S16 in Supplementary Material). Moreover, this technique is also very efficient, inducing transgene expression levels comparable to viral vectors in some cells (Bilal et al., 2015). The use of primary cells derived from patients or healthy donors provides a more accurate model for in vitro and in vivo experiments, and these cells can also be used in cell therapy approaches to treat a large number of diseases. It would be interesting to test this strategy in stem cell differentiation models other than the hematopoietic system such as the central nervous system (Sartore et al., 2011), including models of in vivo differentiation. These cells were plated in methylcellulose-based medium, allowing long-term assessment of GFP expression and differentiation potential. Electroporation | definition of electroporation by - Medical Dictionary There are several ways in which to introduce Cas12a-guide RNA complexes into 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. 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. EnGen Lba Cas12a (Cpf1) (NEB #M0653) is a nuclease that may be used in vivo to create targeted genome modifications. No use, distribution or reproduction is permitted which does not comply with these terms. These authors have contributed equally to this work. 0000006312 00000 n (B) Long-term GFP expression was evaluated until d + 30 post nucleofection with (white bar) or without (black bar) the addition of SB100 transposase (1 g per cuvette). 15, 541555. For CD34+ cells separation, mononuclear cells (MNCs) were isolated from umbilical cord blood after Ficoll density gradient using the same protocol above described. Copyright: 2017 Chicaybam, Barcelos, Peixoto, Carneiro, Limia, Redondo, Lira, Paraguass-Braga, Vasconcelos, Barros and Bonamino. 0000001120 00000 n 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. All cells were seeded in 12-well plates and grown at 37C and 5% CO2. Viruses. 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. 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)]. Gene Ther. Hi, So I am trying to electroporate human dermal fibroblasts using the Lonza 4D. doi:10.1038/mt.2008.6, Yarmush, M. L., Golberg, A., Sera, G., Kotnik, T., and Miklavi, D. (2014). U.S.A. 100, 1422914234. Protocol: Electroporation. MSCs were obtained from healthy donors submitted to surgery for hernia repair at the Clementino Fraga Filho University Hospital. HHS Vulnerability Disclosure, Help 0000022740 00000 n Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments. The ability to introduce DNA, RNA or proteins into cells to alter their genotype or phenotype (a process called transfection) is crucial in a variety of life science applications. Data from electroporation experiments were analyzed by one-way ANOVA followed by Tukeys multiple comparison test using GraphPad Prism 6 software. Please refer to the Lonza 4D-Nucleofector manual for proper usage of the equipment. -. doi:10.1158/1535-7163.MCT-07-0009, Bonamino, M., Serafini, M., DAmico, G., Gaipa, G., Todisco, E., Bernasconi, S., et al. Biol. GFP expression was analyzed until d + 30 for each cell line. Viability data were normalized with viability from non-transfected cells. The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. doi:10.1038/gt.2009.54, Ramanayake, S., Bilmon, I., Bishop, D., Dubosq, M.-C., Blyth, E., Clancy, L., et al. During the incubation, trypsinize the cells, washing once to remove any traces of trypsin. Electroporation of CRISPR/Cas9 cassettes promotes gene editing of PBMCs and 293T cells. Science 341, 1233151. doi:10.1126/science.1233151, PubMed Abstract | CrossRef Full Text | Google Scholar, Aluigi, M., Fogli, M., Curti, A., Isidori, A., Gruppioni, E., Chiodoni, C., et al. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Cells transfected by 93, 896908. electroporation. Manufacture of T cells using the sleeping beauty system to enforce expression of a CD19-specific chimeric antigen receptor. (2015). Front. Clinical Scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. Chem. Colonies were quantified for mock (left) and GFP electroporated (right) cells. Epicentre QuickExtract DNA Extraction Solution (Epicentre #QE09050), 65C for 15 min Electroporation experiments require standard cell culture reagents and instruments appropriate for maintenance of cells. 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. These results probably reflect the observed differences in nucleofection efficiency and proliferation rates among the studied cells. Figure 7. Optimization of the transfection of human THP-1 macrophages by - PubMed U.S.A. 112, 1043710442. Epub 2013 Mar 26. Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B. Mol. 23, 13801390. Low-cost generation of good manufacturing practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. Cancer 4, 551561. Viability (blue bar), GFP expression (green bar), and electroporation score (red bar) were assessed 1 day after nucleofection (d + 1). doi:10.1038/mt.2010.169, Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Optimization of methods for the genetic modification of human T cells. Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program. Various types of transfection methods exist and choosing which approach to use often depends on its suitability to the application in question. doi:10.1016/j.tibtech.2015.06.002, Kowarz, E., Lscher, D., and Marschalek, R. (2015). 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). : MIR 50121). 33, 402416. with 1-mm electroporation cuvettes (Cell . For adherent cells, viability determination was calculated based on the % of the OD obtained in Crystal Violet staining assays at d + 1 or d + 3. Methods 3, 109116. Electroporation using square-wave generating devices, like Lonza's 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. The GFP+ B16F10 cells not only retained GFP expression level, but also kept a constant ratio of GFP+/GFP cells throughout the 15-day period of in vivo tumor development. A., and Bonamino, M. H. (2013). In the following sections, we provide an overview of Nucleofector Technology, and explore what benefits it can bring to your research over traditional electroporation methods. (2011). Methods Mol. Belay E, Dastidar S, VandenDriessche T, Chuah MK. The clinical relevance of cancer cell lines. CGL, CL, FP-B, and ZV performed electroporation and differentiation experiments in CD34+ cells and data analysis and interpretation. Place SOC recovery medium in a 37C water bath. pLR: a lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters. For cell lines not described in this study, Chicabuffers represent a good starting point for the optimization of electroporation protocol and facilitate the genetic modification of cell lines that are not frequently used. We have developed three different Nucleofector Platforms that offer a range of different specifications and units to enable flexible scaling (i.e., Nucleofection of varying numbers and volume of cells depending on the application) as well as transfection of adherent cells. Mesenchymal stem cells do not prevent antibody responses against human -l-iduronidase when used to treat mucopolysaccharidosis type I. PLoS ONE 9:e92420. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Type in Product name, Keyword or Catalog number to see suggestions. doi:10.1038/nmeth846, Vargas, J. E., Salton, G., Sodr de Castro Laino, A., Pires, T. D., Bonamino, M., Lenz, G., et al. After 24 h, the culture medium was changed to eliminate non-adherent cells. Sterile 1X PBS without Ca2+ and Mg2+ 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). LC, CB, BP, MC, PR, and LB performed the electroporation experiments (cell lines, MSCs, and PBMCs) and data analysis and interpretation. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. 369, 134144. Int. QUICKEXRACT is a trademark of Lucigen. 11, 8593. However, the 4D-Nucleofector. 9, 257267. However, these applications often depend on genetic modification, which is usually hard to perform in these cells.
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