electroporation protocol lonza
doi:10.1016/S1525-0016(03)00211-9, Berdien, B., Mock, U., Atanackovic, D., and Fehse, B. Immunol. sharing sensitive information, make sure youre on a federal You have been idle for more than 20 minutes, for your security you have been logged out. Acad. doi:10.1016/j.ymeth.2003.11.009, Griffin, J. L., and Shockcor, J. P. (2004). doi:10.1371/journal.pone.0020667, Satkauskas, S., Ruzgys, P., and Venslauskas, M. S. (2012). Cells transfected by 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. In the case of viral vectors, especially retroviral and lentiviral vectors, there is a wide availability of constructs carrying selectable markers, fluorescent reporters, promoters for different finalities, and cassette configurations, increasing the options of possible cellular manipulations (Szulc et al., 2006; Weber et al., 2008; Vargas et al., 2012). 2022 Dec 2;14(12):2700. doi: 10.3390/pharmaceutics14122700. . Cells were transferred to a sterile 0.2-cm cuvette and electroporated using the reported program (Table 1) of Lonza Nucleofector II electroporation system. Figure 5. (2006). -, Barretina J., Caponigro G., Stransky N., Venkatesan K., Margolin A. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Methods Mol. Furthermore, we showed efficient CRISPR-mediated genome editing of PDCD1 gene in 293T and human PBMCs electroporated using Chicabuffers. 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. The authors also thank all the researchers who provided the cell lines used in this study. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. HEK293FT and PBMCs were electroporated with pX330-PDCD-1 (10 g) and pRGS-CR-target (5 g). J. Pharm. If you don't see your country above, please visit our Unauthorized use of these marks is strictly prohibited. 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. Claudin-3 overexpression increases the malignant potential of colorectal cancer cells: roles of ERK1/2 and PI3K-Akt as modulators of EGFR signaling. Hold at 4C. A live-cell platform to isolate phenotypically defined subpopulations for spatial multi-omic profiling. J. 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. (A) MSCs were electroporated with each one of the seven buffers and the recommended program. A., and Charpentier, E. (2014). The pre-loaded electroporation programs suited for each cell line simplify the experimental setup, and the use of proprietary additives improves the transfection efficiency. 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. Bioeng. After 3 days, the cells were recovered and analyzed by flow cytometry for GFP expression. Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis. doi:10.1002/biot.201400821, Kuystermans, D., and Al-Rubeai, M. (2015). A., Kim S., et al. The origin and cell culture conditions for each cell line are described in Table S1 in Supplementary Material. These benefits can facilitate numerous applications, such as therapeutic gene knock-down via RNAi2 or CRISPR3 and the generation of induced pluripotent stem cells4 or CAR-T5, among many others. Cancer 4, 551561. After 15 days, we excised the tumor and plated the cells in 25 cm2 culture flasks. Preprint. The electroporation protocol for each cell line is summarized in Table 1. 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. GFP expression was analyzed until d + 30 for each cell line. : MIR 50121). 0000022467 00000 n 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). U.S.A. 112, 1043710442. Non-viral vectors for gene-based therapy. The hematopoietic progenitor CD34+ cells were evaluated for purity by staining with anti-CD34-PE (clone 581, BD Biosciences). Numbers depict the percentages of cells in each gate. No use, distribution or reproduction is permitted which does not comply with these terms. Nat. J. Natl. Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation. Numbers depict the percentage of cells inside each gate. Determine the cell number and viability using a hemocytometer. Synthetic gene transfer vectors II: back to the future. CD34; CRISPR; GFP; MSC; PD-1; T lymphocyte; cell line; electroporation; transposon. Science 341, 1233151.10.1126/science.1233151 Significant differences (p < 0.05) are depicted in the table next to each graph, with each color denoting one parameter. Generic cuvettes were used for all the electroporations (Mirus Biotech, Madison, WI, USA cat. Section 1 Background. Natl. Calculate the volume of Nucleofector Solution you will need to re-suspend the cells (10.5 l per transfection). Colonies were quantified for mock (left) and GFP electroporated (right) cells. Biol. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figure 2. Chem. 0000007521 00000 n Acc. An official website of the United States government. To assess viability of adherent cell lines, cells were plated in triplicate in 96-well microtiter plates immediately after electroporation. doi:10.1038/icb.2015.59, Blower, P. E., Verducci, J. S., Lin, S., Zhou, J., Chung, J.-H., Dai, Z., et al. Cancer Gene Ther. 45, 980984.10.1021/ar200213g Please login or create an account. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on ice. (2012). Federal government websites often end in .gov or .mil. The new constructs described in this report are available at Addgene. Add 75 l of media to the cells in the cuvette, pipetting gently. 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). Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses. electroporation and Nucleofection Experiment. Genet. Belay E, Dastidar S, VandenDriessche T, Chuah MK. Analysis of editing can be done following the protocol detailed in the EnGen Mutation Detection Kit (NEB #. 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. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. 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. Flexible Scaling and Adherent Electroporation via Nucleofection. In this system, GFP expression can be restored by CRISPR-mediated NHEJ repair (Kim et al., 2011), leading to restoration of the reading frame in nearly 1/3 of the editions. New non-viral method for gene transfer into primary cells. Data from electroporation experiments were analyzed by one-way ANOVA followed by Tukeys multiple comparison test using GraphPad Prism 6 software. Electroporation Protocol (C2986) | NEB Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. Sci. EnGen Lba Cas12a (Cpf1) (NEB #M0653) (2007). Biomed. Cell viability was evaluated after 24 h, and cell expansion was analyzed at day + 1 by crystal violet. Our solution is an improved electroporation technology, the Nucleofector Technology, originally introduced into the market by legacy Amaxa in 2001. Sleeping beauty-based GFP gene transfer to human cord blood CD34+ cells. Manufacture of T cells using the sleeping beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Viruses. doi:10.1038/gt.2014.26, Bilal, M. Y., Vacaflores, A., and Houtman, J. C. (2015). The authors thank Sang Wang Han (UNIFESPBrazil) for pT2-GFP and SB100 plasmids, Richard Morgan (NIH) for pT3-GFP plasmid, and Amilcar Tanuri (UFRJ) for pRGS-CR plasmid. To save your cart and view previous orders, sign in to your NEB account. Hollis RP, Nightingale SJ, Wang X, Pepper KA, Yu XJ, Barsky L, Crooks GM, Kohn DB. Select the pre-optimized program for HEK293 cells (CM-130) and electroporate the cells. Incubate the cells in a humidified 37C, 5% CO. Gently aspirate the media from the cells and wash twice with 250 l 1X PBS. Long-term transgene expression in electroporated cell lines using sleeping beauty system. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. B16F10 cells were electroporated with 4 g of pT3-NEO-EF1a-GFP and 1 g of SB100 in buffer 1S, program P-020 of Lonza Nucleofactor II. (2004). Mol. 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). 427, 320. Tumors were extracted 14 days post injection (dpi); cells were passed in vitro for 1 week and GFP expression analyzed by FACS. Front. Clinical Scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. 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. Stem Cells 24, 454461.10.1634/stemcells.2005-0198 Such pores enable commodities such as DNA, PROTEINS and ATP to pass into the cell, before the pores are repaired. Genetic modification of cells is a cumbersome and expensive process, often involving the use of viral vectors to achieve high efficiency transgene expression. Recently, the development of gene editing tools like TALENs and CRISPRs provided a more precise control of gene insertion or deletion, extending the possible genomic manipulations (Kim and Kim, 2014). Exp Hematol. Cytotherapy 17, 12511267. doi:10.1038/nrc1390, Hamid, O., Robert, C., Daud, A., Hodi, F. S., Hwu, W.-J., Kefford, R., et al. Conditions can be seamlessly transferred between these two vessel types. National Library of Medicine 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. Each condition was plated in a 6-well plate. 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. doi:10.1016/j.ijpharm.2011.07.013, Weber, K., Bartsch, U., Stocking, C., and Fehse, B. (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. Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Ther. 0000003955 00000 n Accessibility (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). A multicolor panel of novel lentiviral gene ontology (LeGO) vectors for functional gene analysis. The cells were concentrated in 300 L and then added in 1.1 concentrated 3 mL Methocult H4034 (Stem Cell Technologies Inc., Vancouver, BC, Canada), then seeded two wells of a six-well plates, 1.1 mL/well. Cell lines were electroporated with pT2-GFP (4 g) using each one of the seven buffers and the recommended program. Epub 2022 Nov 3. Data are shown as mean SD from three experiments performed in duplicate and were further analyzed using one-way ANOVA with Tukeys multiple comparisons test. Nat. Prepare a culture plate with the appropriate volume of media. The Nucleofector Technology offers a higher transfection efficiency than other non-viral transfection methods (including traditional electroporation1) along with other significant advantages outlined below. bioRxiv. The EF1a-GFP cassette was isolated from the plasmid pRRLsin.PPTs.EF1a.GFPpre (Bonamino et al., 2004) (provided by Dr. Didier Trono, EPFL, Switzerland) after digestion with ClaI/BstBI and inserted in pT3-NEO previously digested with ClaI. As showed in Figure 3, the addition of SB100 induced a higher percentage of GFP-positive cells after 30 days of culture when compared with control cells, strongly suggesting that integration of the transgene has occurred. GFP expression after electroporation of representative cell lines. 12 0 obj <> endobj xref 12 29 0000000016 00000 n doi:10.1038/mt.2008.6, Yarmush, M. L., Golberg, A., Sera, G., Kotnik, T., and Miklavi, D. (2014). MeSH Moreover, the use of electroporation is associated with extensive testing of electric parameters (pulse amplitude, volts) in order to optimize the protocol. 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. 0000001372 00000 n To achieve efficient gene editing of target cells, Cas9 nuclease and the gRNA must be expressed in the cell, ideally in a transient fashion. Stable gene expression is often required in the experimental setting, allowing the generation of subclones with overexpression or silencing of a gene of interest. The following additional materials are required but not supplied: horizondiscovery.com Electroporation instrument Electroporation reagents (buffer, cuvettes, transfer pipettes) Proc. 7AAD staining (eBioscience cat. Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays. doi:10.1371/journal.pone.0074994, Doudna, J. Transfection efficiency was monitored by flow cytometry after 24 hours. HEL was electroporated using buffer 2S and program X-005 and NIH3T3 using buffer 1SM and program U-030. It is a phenomenon in which the permeability of cell membrane to ions and macromolecules is increased by exposing the cell to high voltage electric pulses. (2008). EnGen Lba Cas12a (Cpf1) (NEB #M0653) is a nuclease that may be used in vivo to create targeted genome modifications. Ther. Importantly, after 24 h of electroporation, the cells showed a good viability (Figure 2), allowing expansion and recovery from the nucleofection. Aiuti, A., Biasco, L., Scaramuzza, S., Ferrua, F., Cicalese, M. P., Baricordi, C., et al. 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. All cells were seeded in 12-well plates and grown at 37C and 5% CO2. Nonviral gene delivery with the sleeping beauty transposon system. (2015), showing that different gRNAs can be used to efficiently disrupt the PDCD1 gene sequence. 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. The single plasmid approach for PBMC edition is also simpler to assemble than the recently reported Cas9+ gRNA ribonucleoproteins (Schumann et al., 2015), showing that our extremely simple protocol can be used to edit cell genomes. 15, 541555. doi:10.1038/gt.2009.54, Ramanayake, S., Bilmon, I., Bishop, D., Dubosq, M.-C., Blyth, E., Clancy, L., et al. NUCLEOFECTOR is a registered trademark of Lonza Group Ltd. The utilization of CD34+ cells was also approved by INCAs Ethics Committee. Hi, So I am trying to electroporate human dermal fibroblasts using the Lonza 4D. During the incubation, trypsinize the cells, washing once to remove any traces of trypsin. 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. The GFP expression can be restored by CRISPR-mediated non-homologous end joining (NHEJ) repair. Electroporation - an overview | ScienceDirect Topics LC and MB took part in the conception and design of the study, data interpretation, and manuscript writing. Data were analyzed using the FlowJo software (Tree Star). 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). Amaxa General Nucleofection Protocol - Lonza Mol. Biol. For further technical assistance on using the Nucleofector Technology for CRISPR-based genome editing, contact Lonza Scientific Support. NUCLEOCUVETTE is a trademark of Lonza Group Ltd. An efficient low cost method for gene transfer to T lymphocytes. Unable to load your collection due to an error, Unable to load your delegates due to an error. In a previous work, our group developed in house electroporation buffers (termed Chicabuffers) that had comparable efficiency with Lonzas buffers for the transfection of the human T cell line Jurkat and primary T lymphocytes from mouse and human origin (Chicaybam et al., 2013). Electroporation and Nucleofector Technology | Lonza Ther. 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). 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. Electroporation experiments require standard cell culture reagents and instruments appropriate for maintenance of cells. Cell Biol. The modular 4D-Nucleofector System helps you scale up from low to high-volume transfection or higher throughput without having to re-optimize. Ther. doi:10.1038/ng.343, Mir, L. M. (2014). Various types of transfection methods exist and choosing which approach to use often depends on its suitability to the application in question. CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo. -, Aluigi M., Fogli M., Curti A., Isidori A., Gruppioni E., Chiodoni C., et al. Nat. Plasmid 68, 179185. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The new vectors developed and validated in the present report can improve flexibility and increase the applicability of this system, promoting accessible and efficient transgene integration into different cell types.
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