2013;8(3):e60298. We selected cells lines representing models for hematopoietic neoplasias (HEL, K562, P815, Nalm-6, and Jurkat cell lines) and different solid tumor-derived cell lines (A-549, B16-F10, HeLa, MCF-7, MDA-MB-231). Adherent cells were allowed to adhere overnight after electroporation, and non-adherent/dead cells were discarded before FACS analysis. Cells were centrifuged for 20 min at 890 g (slow acceleration/deceleration off), washed three times with PBS, and used for nucleofection. After Ficoll gradient purification, PBMCs (107 cells) were electroporated with pRGS-CR-target (10 g) and 1050 pmol of FITC labeled RNA (Invitrogen) in Chicabuffer 3 P and U-014 Nucleofector IIb program. 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. (2003). The 4D-Nucleofector X Unit features positions for both 20 L Nucleocuvette Strips and 100 L single Nucleocuvette Vessels. CFU-E, colony-forming unit-erythroid; BFU-E, burst-forming unit-erythroid; CFU-G/M/GM, colony-forming unit-granulocyte/monocyte/; CFU-GEMM, colony-forming unit-granulocyteerythrocytemonocytemegakaryocyte. Figure 6. To achieve efficient gene editing of target cells, Cas9 nuclease and the gRNA must be expressed in the cell, ideally in a transient fashion. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee. 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. 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. You have been idle for more than 20 minutes, for your security you have been logged out. Optimized sleeping beauty transposons rapidly generate stable transgenic cell lines. Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B. Mol. Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. DMEM with Glutamax (or appropriate growth medium) with 10% FBS CGL, CL, FP-B, and ZV performed electroporation and differentiation experiments in CD34+ cells and data analysis and interpretation. (2010). During the incubation, trypsinize the cells, washing once to remove any traces of trypsin. The crystal violet incorporation assay was performed by fixing the cells with ethanol for 10 min, followed by staining them with 0.05% crystal violet in 20% ethanol for 10 min and solubilization with methanol as reported (Faget et al., 2012). For CD34+ cells separation, mononuclear cells (MNCs) were isolated from umbilical cord blood after Ficoll density gradient using the same protocol above described. 0000001120 00000 n These authors have contributed equally to this work. In May 2023, Frontiers adopted a new reporting platform to be Counter 5 compliant, in line with industry standards. Keywords: Biomed. -, Behr J.-P. (2012). Table 1. (C) Summarized results obtained for 293T cells and PBMCs, showing the number of colonies sequenced and the percentage of indels detected. (2009). 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. 1 Maasho et al 2004 Journal of Immunological Methods, 2 Marques and Williams, 2005 Nature Biotechnology. 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. Eng. 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. 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). This setting clearly allows efficient co-electroporation of plasmid DNA and short RNA, opening the possibility of combining siRNA and transgene expression or even multiple gRNAs and Cas9 expressing plasmids for gene editing. A guide to genome engineering with programmable nucleases. Transfected samples may be plated in any number of replicates. 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. Trends Biotechnol. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Cancer 4, 551561. The HT Nucleofector System is an independent platform offering high-throughput transfection in 384-well format. Representative plots of a high electroporation score cell line (HEL) and a low score cell line (NIH3T3). (2009). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Transfection of adherent and suspended cells by calcium phosphate. Synthetic gene transfer vectors II: back to the future. 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. From electroporated cells under the same condition, up to 14.8% co-expressed the reporter plasmid (encoding RFP) and the labeled short RNA (Figure S19C Supplementary Material). Belay E, Dastidar S, VandenDriessche T, Chuah MK. A versatile tool for conditional gene expression and knockdown. doi:10.1002/biot.201400821, Kuystermans, D., and Al-Rubeai, M. (2015). Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays. The authors also thank all the researchers who provided the cell lines used in this study. We also offer solutions for automation, site-directed mutagenesis, as well as your favorite restriction enzyme, ligase or competent cell products. Mol. Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses. 18, 12001209. (B) Electroporated cells (2 103 per well) were plated in methylcellulose media and allowed to differentiate for 3 weeks. Biotechnol., 23 January 2017, https://www.frontiersin.org/article/10.3389/fbioe.2016.00099/full#supplementary-material, Creative Commons Attribution License (CC BY). Bioeng. 00-6693) was performed immediately before FACS acquisition following manufacturer instructions. Each condition was plated in a 6-well plate. Gene editions were evaluated by GFP+/RFP+ ratio after 24 h by flow cytometry. The Lonza 4D-Nucleofector platform has multiple electroporation volume and vessel options. doi:10.1016/j.ymeth.2003.11.011, Kim, H., and Kim, J.-S. (2014). (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. 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. Pre-warm selective plates at 37C for 1 hour. with 1-mm electroporation cuvettes (Cell . Acad. 0000009213 00000 n The pre-loaded electroporation programs suited for each cell line simplify the experimental setup, and the use of proprietary additives improves the transfection efficiency. a method in which CELLS are subjected to an electrical impulse that leads to the temporary formation of pores in the cell MEMBRANE. Tumors were extracted 14 days post injection (dpi); cells were passed in vitro for 1 week and GFP expression analyzed by FACS. MSCs were obtained from healthy donors submitted to surgery for hernia repair at the Clementino Fraga Filho University Hospital. 45, 980984. The pT2-GFP and SB100X (Mts et al., 2009) constructs were kindly provided by Dr. Copyright: 2017 Chicaybam, Barcelos, Peixoto, Carneiro, Limia, Redondo, Lira, Paraguass-Braga, Vasconcelos, Barros and Bonamino. Only registered User can view this video. After 15 days, we excised the tumor and plated the cells in 25 cm2 culture flasks. National Library of Medicine For non-adherent cell lines, viability determination was based on trypan blue exclusion and/or determination of the % of cells displaying viable cell FSC vs. SSC parameters by flow cytometry analysis on cells negative after 7AAD staining. Rev. Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication. 18, 18961906. 0000007521 00000 n Proc. We also summarize the transfection capabilities and flexible scaling offered by our various Nucleofector Platforms, to help you choose which one is best suited to your specific application. Colonies were quantified for mock (left) and GFP electroporated (right) cells. Add 10.5 l of cells to each 14.5 l RNP reaction. 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. 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). Manufacture of T cells using the sleeping beauty system to enforce expression of a CD19-specific chimeric antigen receptor. doi:10.1158/1535-7163.MCT-09-0743, Peng, P. D., Cohen, C. J., Yang, S., Hsu, C., Jones, S., Zhao, Y., et al. Excellent preservation of the physiological status and viability of transfected cells. QUICKEXRACT is a trademark of Lucigen. It is therefore not surprising that Nucleofector Technology is now used in many different lines of research, including functional and structural genomics, drug discovery, and gene and cell therapy. Methods 33, 136143. The medium was replaced by complete RPMI medium the following day, and cells were maintained as described previously. Nat. The utilization of CD34+ cells was also approved by INCAs Ethics Committee. 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. For long-term experiments, 1 g of SB100 was added. LC, CB, BP, MC, PR, and LB performed the electroporation experiments (cell lines, MSCs, and PBMCs) and data analysis and interpretation. Ther. Generic cuvettes were used for all the electroporations (Mirus Biotech, Madison, WI, USA cat. In addition, cells with clear therapeutic potential, such as T lymphocytes (Chicaybam et al., 2013) and MSCs (this report) could be stably modified using a combination of Chicabuffer, SB, and electroporation. Low-cost generation of good manufacturing practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. Desired culture plate, DNA Extraction and Genome Editing Analysis, EnGen Mutation Detection Kit (NEB #E3321) Here we present a method for the introduction of Cas12a RNPs into HEK293 FT cells using the Lonza 4D-Nucleofector Electroporation System. EnGen Lba Cas12a (Cpf1) (NEB #M0653) MicroRNA expression profiles for the NCI-60 cancer cell panel. The NucleofectorTechnology uses a specific combination of optimized electrical parameters and cell type-specific solutions which enables transfer of a molecule directly into the cells nucleus. The medium was changed every 3 days and the antibiotic added. by incubation on ice 1.2 Inoculate 500 ml YT with 5 ml of an overnight culture of the bacteria in a large Erlenmeyer flask and strongly shake them at 37C unti. Genet. The Lonza 4D-Nucleofector platform has multiple electroporation volume and vessel options. J. The meaning of ELECTROPORATION is the application of an electric current to a living surface (such as the skin or a cell membrane) in order to open pores or channels through which something (such as a drug or DNA) may pass. 16, 295320. 2022 Oct 24;10(11):2687. doi: 10.3390/biomedicines10112687. Moreover, cell lines are central players in the biotechnology industry, being used in the production of biopharmaceuticals like antibodies, hormones, and bioactive proteins in general (Kuystermans and Al-Rubeai, 2015). doi:10.1007/978-1-4614-9632-8_1, Nishizuka, S., Charboneau, L., Young, L., Major, S., Reinhold, W. C., Waltham, M., et al. The new frontier of genome engineering with CRISPR-Cas9. 23, 13801390. Front. The origin and cell culture conditions for each cell line are described in Table S1 in Supplementary Material. When using SB100, long-term expression of GFP using this buffer was seen in 12% of cells (Figure 5B). U.S.A. 112, 1043710442. Add 50 l of Epicentre QuickExtract DNA Extraction Solution and shake/vortex for 5 minutes. Various types of transfection methods exist and choosing which approach to use often depends on its suitability to the application in question. Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. All the authors read and approved the final manuscript. Transfection efficiency was monitored by flow cytometry after 24 hours. Chicabuffers showed to work for all the cells tested with most of the samples showing interchangeable results among the different buffers and only few exceptions where one of the buffers performed poorly or GFP expression was improved at the expense of cell viability, such as for buffer 3P in Jurkat cells. Gene Ther. The hematopoietic progenitor CD34+ cells were evaluated for purity by staining with anti-CD34-PE (clone 581, BD Biosciences). J. Pharm. 0000005643 00000 n 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. 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. Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP) into adherent cells using the Lonza 4D-Nucleofector, Next Generation Sequencing Library Preparation, DNA Assembly, Cloning and Mutagenesis Kits, Supporting Infectious Disease Research & Development, We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Ther. Cell lines were electroporated with pT2-GFP (4 g) using each one of the seven buffers and the recommended program. A., Kim S., 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. %PDF-1.6 % The following additional materials are required but not supplied: horizondiscovery.com Electroporation instrument Electroporation reagents (buffer, cuvettes, transfer pipettes) Trypsin to release cells Suspension cell lines showed the best results regarding GFP expression, in which values above 60% were recurrently obtained. 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. Significant differences (p < 0.05) are depicted in the table next to each graph, with each color denoting one parameter. 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. Find out more by visiting the HT Nucleofector System product pageor watching our video tutorial. 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. Sci. 10, 647653. After 3 weeks, GFP+CD34+ cells were able to differentiate to erythroid, granulocytic, and myeloid lineages (Figure 6B), showing that the insertion of the transgene did not affect the stemness of the cells and that differentiated cells display high GFP expression (Figure S17 in Supplementary Mateiral). Transfer the solution to a PCR plate or tubes and place in a thermocycler, running the following program. 2019 Apr;30(4):511-522. doi: 10.1089/hum.2018.218. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. Calculate the number of cells you will need for the entire experiment (1-2 x 10. Careers. Our solution is an improved electroporation technology, the Nucleofector Technology, originally introduced into the market by legacy Amaxa in 2001. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. 11, 8593. Genetic modification of cells is a cumbersome and expensive process, often involving the use of viral vectors to achieve high efficiency transgene expression. Determine the cell number and viability using a hemocytometer. U.S.A. 100, 1422914234. 0000022740 00000 n . Transfer the desired number of cells to wells in the culture plate. 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. Biopharmaceutical Products from Animal Cell Culture, in Animal Cell Culture Cell Engineering. 9, 257267. Methods 8, 941943. Actual pmols of RNA and protein may be optimized. 427, 320. Acc. Cells were electroporated at passage 3. 0000004777 00000 n 0000001372 00000 n 12, 275286. Summarized electroporation conditions for each cell line (based in Figure 2; d1 after electroporation). Surrogate reporters for enrichment of cells with nuclease-induced mutations. Transposon-mediated gene transfer into adult and induced pluripotent stem cells. (2015). doi:10.1371/journal.pone.0074994, Doudna, J. Bethesda, MD 20894, Web Policies Res. doi:10.1016/S1525-0016(03)00211-9, Berdien, B., Mock, U., Atanackovic, D., and Fehse, B. HEL was electroporated using buffer 2S and program X-005 and NIH3T3 using buffer 1SM and program U-030. 0000003101 00000 n E. coli can be either bought from different sources or prepared as described below: 1.1 Pre-cool approx. CD34+ cells were isolated from MNCs using CD34 MicroBead Kit (Miltenyi Biotech) following the manufacturers instructions. Keep up to speed on the latest scientific developments, events, tips and tools from Lonza. Khatib TO, Amanso AM, Pedro B, Knippler CM, Summerbell ER, Zohbi NM, Konen JM, Mouw JK, Marcus AI. 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. Ther. Competitor B electroporation: 25 mV, 96 F. Place SOC recovery medium in a 37C water bath. Molecular evolution of a novel hyperactive sleeping beauty transposase enables robust stable gene transfer in vertebrates. -, Barretina J., Caponigro G., Stransky N., Venkatesan K., Margolin A. 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. 2023 Mar 1:2023.02.28.530493. doi: 10.1101/2023.02.28.530493. An official website of the United States government. Furthermore, the protocol described for the co-electroporation of short RNAs and plasmids carrying GFP+ Cas9 can be exploited for multiple loci editing in PBMCs, opening the possibility of targeting simultaneously several genes of interest. This illustrates that privileging GFP expression, for instance, can be detrimental to cell viability, opening room to further improvements in the electroporation protocol or buffer formulations. (2007). Bioeng. Hollis RP, Nightingale SJ, Wang X, Pepper KA, Yu XJ, Barsky L, Crooks GM, Kohn DB. Mol. 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. For efficient electroporation, detachment of adherent cells is necessary. Set up the RNP formation reaction as follows. 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). doi:10.1038/nature11003, Beane, J. D., Lee, G., Zheng, Z., Mendel, M., Abate-Daga, D., Bharathan, M., et al. Chicaybam L, Sodre AL, Curzio BA, Bonamino MH. doi:10.1038/nmeth846, Vargas, J. E., Salton, G., Sodr de Castro Laino, A., Pires, T. D., Bonamino, M., Lenz, G., et al. Mesenchymal stem cells were isolated from abdominal subcutaneous adipose tissue fragments obtained from healthy donors submitted to surgery for hernia repair at the Clementino Fraga Filho University Hospital. Rev. Add the entire 25 l of RNP/cell mixture to the cuvette strips. 0000008466 00000 n In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. 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). Biotechnol. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee.
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