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. Excellent preservation of the physiological status and viability of transfected cells. J. Med. doi:10.1038/icb.2015.59, Blower, P. E., Verducci, J. S., Lin, S., Zhou, J., Chung, J.-H., Dai, Z., et al. Transfected samples may be plated in any number of replicates. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. doi:10.1038/mt.2008.6, Yarmush, M. L., Golberg, A., Sera, G., Kotnik, T., and Miklavi, D. (2014). Claudin-3 overexpression increases the malignant potential of colorectal cancer cells: roles of ERK1/2 and PI3K-Akt as modulators of EGFR signaling. Viability data were normalized with viability from non-transfected cells. Ther. According to the severity of damage on cell . 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. 6, 14831491. For long-term experiments, 1 g of SB100 was added. Our solution is an improved electroporation technology, the Nucleofector Technology, originally introduced into the market by legacy Amaxa in 2001. 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. Biotechnol. This method uses a guide RNA to protein ratio of 10:1. 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. doi:10.1016/j.plasmid.2012.06.001, Wang, T., Upponi, J. R., and Torchilin, V. P. (2012). Genet. Transfection of hard-to-transfect cells, including primary cells, stem cells, neurons and cell lines, as well as cells in adherence. Immunol. Biol. Add the entire 25 l of RNP/cell mixture to the cuvette strips. (2013). Calculate the number of cells you will need for the entire experiment (1-2 x 10. (2011). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. 12 0 obj <> endobj xref 12 29 0000000016 00000 n MSCs were obtained from healthy donors submitted to surgery for hernia repair at the Clementino Fraga Filho University Hospital. Select the pre-optimized program for HEK293 cells (CM-130) and electroporate the cells. Designing a single plasmid encoding Cas9+ gRNA is simpler than constructing zinc finger nuclease (Beane et al., 2015) or TALEN (Berdien et al., 2014)-based cassettes. The electroporation score calculated for every cell line is a general guide for electroporation efficiency comparison, and the buffer choice can be adapted to the need of the planned experiment (higher GFP expression or cell viability), allowing the researcher to experiment with different transgene expression levels. doi:10.1038/mt.2010.169, Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. No use, distribution or reproduction is permitted which does not comply with these terms. See how transfection with Nucleofection Technology is different from conventional electroporation. Figure 2. Optimization of methods for the genetic modification of human T cells. These authors have contributed equally to this work. Mol. For HEL, on day 1 after nucleofection, cells were stained with 7AAD (left column of graphs) and GFP expression was analyzed on 7AAD negative population (right column). Sci. FOIA GFP and red fluorescent protein expression was analyzed 24 h later. MeSH To evaluate the performance of Chicabuffers in the gene transfer to these cells, we isolated adipose tissue derived MSCs and cord blood purified CD34+ hematopoietic stem cells and electroporated the cells with the plasmids pT2-GFP and SB100. Bethesda, MD 20894, Web Policies All plasmids were isolated using Qiamp Maxi prep kit from Qiagen (Germany) and quantified using a Nanodrop spectrophotometer. Prepare a culture plate with the appropriate volume of media. The clinical relevance of cancer cell lines. 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. The results of gene editing experiments in 293T and PBMCs are summarized in Figure 7C. 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). Nat. Desired culture plate, DNA Extraction and Genome Editing Analysis, EnGen Mutation Detection Kit (NEB #E3321) Acad. 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. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee. Preprint. 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. Analysis of editing can be done following the protocol detailed in the EnGen Mutation Detection Kit (NEB #. The patients provided written informed consent, and the study was approved by the Hospital Research Ethics Committee. Chicaybam L, Sodre AL, Curzio BA, Bonamino MH. (2006). 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. However, costs associated with the acquisition of nucleofection kits, especially if used in a routine basis, might hamper the use of this technology in some laboratories or impair large-scale experiments. 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). doi:10.1007/978-1-4614-9632-8_1, Nishizuka, S., Charboneau, L., Young, L., Major, S., Reinhold, W. C., Waltham, M., et al. Protocol for Electroporation of EnGen SpyCas9 NLS, - NEB (2015). Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. (2006). Significant differences (p < 0.05) are depicted in the table next to each graph, with each color denoting one parameter. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Cells were transferred to a sterile 0.2-cm cuvette and electroporated using the reported program (Table 1) of Lonza Nucleofector II electroporation system. electroporation. Gently mix the Nucleofector solution, EnGen Lba Cas12a, and gRNA and incubate at room temperature for 20 minutes. 23, 13801390.10.1038/mt.2015.71 Transfection of a wide range of substrates, including DNA, mRNA, miRNA, siRNA, peptides or proteins. CGL, CL, FP-B, and ZV performed electroporation and differentiation experiments in CD34+ cells and data analysis and interpretation. Hi, So I am trying to electroporate human dermal fibroblasts using the Lonza 4D. 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. Various types of transfection methods exist and choosing which approach to use often depends on its suitability to the application in question. Figure 1. Lonza 4D-Nucleofector X-Unit A., Kim S., et al. Accessibility 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. 15, 541555. PLoS ONE 7:e47868. TheraPEAK Nucleofector Products for the 4D-Nucleofector LV Unit help you expedite translating your non-viral gene modification step into a GMP process. Gene editions were evaluated by GFP+/RFP+ ratio after 24 h by flow cytometry. Keep up to speed on the latest scientific developments, events, tips and tools from Lonza. doi:10.1038/ng.343, Mir, L. M. (2014). Electroporation-based applications in biotechnology. (2010). Acc. Bioeng. For fast and easy enrichment of GFP-positive cells, we constructed a bidirectional vector encoding GFP and G418 resistance in the backbone of pT3 transposon, named pT3-Neo-EF1a-GFP. After selection with antibiotic or not, we injected 5 105 cells in the left flank of C57BL/6 mice. (C) 5 105 B16F10 cells submitted or not to selection with G418 were injected in the left flank of C57Bl/6 mice. We selected 14 cell lines of mouse and human origin and primary human cells [MSC, peripheral blood mononuclear cells (PBMCs), and cord blood CD34+ cells], showing that these buffers yield high transfection efficiencies and are a viable option for genetic modification using the Nucleofector IIb electroporator. However, these applications often depend on genetic modification, which is usually hard to perform in these cells. A density gradient centrifugation using Ficoll-Hypaque-1077 was performed. Request your demo appointment and one of our specialists will contact you shortly to schedule your personal meeting. Curr Gene Ther. doi:10.1002/biot.201400821, Kuystermans, D., and Al-Rubeai, M. (2015). Bookshelf QUICKEXRACT is a trademark of Lucigen. Science 337, 816821. For CD34+ cells separation, mononuclear cells (MNCs) were isolated from umbilical cord blood after Ficoll density gradient using the same protocol above described. 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. Adherent cells were allowed to adhere overnight after electroporation, and non-adherent/dead cells were discarded before FACS analysis. 600 ml sterile water and 10 ml sterile water with 10% glycerol to max. Sleeping beauty-based GFP gene transfer to adipose tissue derived human mesenchymal stem cells (MSCs). doi:10.1038/mt.2010.47, Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., et al. Methods to deliver foreign genetic material (DNA or RNA) usually rely in nonviral or viral vectors, with the former being preferred because of increased biosafety, easier production, and faster translation. doi:10.1038/nrg3763, Keywords: electroporation, cell line, MSC, T lymphocyte, CD34, transposon, CRISPR, PD-1, GFP, Citation: Chicaybam L, Barcelos C, Peixoto B, Carneiro M, Limia CG, Redondo P, Lira C, Paraguass-Braga F, Vasconcelos ZFMD, Barros L and Bonamino MH (2017) An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells. doi:10.1371/journal.pone.0092420, Mts, L., Chuah, M. K. L., Belay, E., Jerchow, B., Manoj, N., Acosta-Sanchez, A., et al. The electroporation protocol for each cell line is summarized in Table 1. The pT3-GFP plasmid (Peng et al., 2009) was kindly provided by Dr. Richard Morgan (Surgery BranchNCI). Mol. Please enable it to take advantage of the complete set of features! Colonies were quantified for mock (left) and GFP electroporated (right) cells. This plasmid was validated in G418 resistance assays using B16F10 cells (data not shown). (2012). 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). 0000002461 00000 n 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)]. 0000009772 00000 n Nucleofection Experiment: Nucleofector Solution V; Program O-017. Amplification of the target region was performed by PCR using the forward 5-CCCCAGCAGAGACTTCTCAA and the reverse 5-AGGACCGGCTCAGCTCAC primers. Two experiments were done for each cell. doi:10.1016/S1525-0016(03)00211-9, Berdien, B., Mock, U., Atanackovic, D., and Fehse, B. 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. 0000003101 00000 n 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. 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/nrc1390, Hamid, O., Robert, C., Daud, A., Hodi, F. S., Hwu, W.-J., Kefford, R., et al. Furthermore, we showed efficient CRISPR-mediated genome editing of PDCD1 gene in 293T and human PBMCs electroporated using Chicabuffers. Transfection of adherent and suspended cells by calcium phosphate. PMC U.S.A. 112, 1043710442. Comparative analysis of transposable element vector systems in human cells. (2013). Data were analyzed using the FlowJo software (Tree Star). Figure 7. Integrative analysis of proteomic signatures, mutations, and drug responsiveness in the NCI 60 cancer cell line set. Nature 483, 603607.10.1038/nature11003 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. Nonviral gene delivery with the sleeping beauty transposon system. Synthetic gene transfer vectors II: back to the future. doi:10.1126/science.1225829, Jordan, M., and Wurm, F. (2004). To characterize indels at PDCD1 locus, genomic DNA of gene edited cells was isolated by phenolchloroform. Add 75 l of media to the cells in the cuvette, pipetting gently. Cell lines are valuable tools for research development, constituting one of the pillars of experimental biology. 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. Mol. Chem. For the validation of gRNA, we used plasmid pRGS-CR-PDCD1, which has the PDCD1 target sequence cloned between a RFP and a GFP, resulting in an out-of-frame GFP. Add 50 l of Epicentre QuickExtract DNA Extraction Solution and shake/vortex for 5 minutes. The monoculture of cord-blood-derived CD34. Federal government websites often end in .gov or .mil. 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. Tap lightly to ensure the liquid is on the bottom and any air bubbles have been released. doi:10.1093/jnci/djt007, Grabundzija, I., Irgang, M., Mts, L., Belay, E., Matrai, J., Gogol-Dring, A., et al. The combination of transposon, Chicabuffers, and electroporation, as described here, represents a straightforward approach for transient gene expression and permanent gene modification of cell lines and human primary cells. Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses. Optimization of the transfection of human THP-1 macrophages by - PubMed A., and Charpentier, E. (2012). 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. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. When you are looking to clone with confidence, think of NEB. The Lonza 4D-Nucleofector platform has multiple electroporation volume and vessel options. After 3 days, the cells were recovered and analyzed by flow cytometry for GFP expression. This protocol is written for use with the 16-well Nucleocuvette Strips. Summarized electroporation conditions for each cell line (based in Figure 2; d1 after electroporation). doi:10.1634/stemcells.2005-0198, Barretina, J., Caponigro, G., Stransky, N., Venkatesan, K., Margolin, A. MicroRNA expression profiles for the NCI-60 cancer cell panel. Seed the cells so that they will be around 70-90% confluent on the day of transfection. 0000004503 00000 n Does anyone have an optimized protocol for electroporation human dermal Proc. Sci. 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). After transfection, cells were gently resuspended in 1 mL of pre-warmed RPMI medium supplemented only with 2mM l-Glutamine and 20% FCS. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. For further technical assistance on using the Nucleofector Technology for CRISPR-based genome editing, contact Lonza Scientific Support. 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. 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. 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. In order to evaluate if Chicabuffers could be used with this system, 1 g of SB100 (encoding a hyperactive version of the SB transposase) was electroporated with 4 g of pT2-GFP and GFP expression was followed for 30 days. Khanal S, Cao D, Zhang J, Zhang Y, Schank M, Dang X, Nguyen LNT, Wu XY, Jiang Y, Ning S, Zhao J, Wang L, Gazzar ME, Moorman JP, Yao ZQ. Chicaybam L, Abdo L, Carneiro M, Peixoto B, Viegas M, de Sousa P, Fornazin MC, Spago MC, Albertoni Laranjeira AB, de Campos-Lima PO, Nowill A, Barros LRC, Bonamino MH. (2008). Aiuti, A., Biasco, L., Scaramuzza, S., Ferrua, F., Cicalese, M. P., Baricordi, C., et al. (2015). The Supplementary Material for this article can be found online at https://www.frontiersin.org/article/10.3389/fbioe.2016.00099/full#supplementary-material. %PDF-1.6 % After G418 selection and withdrawal, GFP expression remained stable in NIH3T3 cells for 15 days (Figure S15 in Supplementary Material). doi:10.1073/pnas.1512503112, Singh, H., Moyes, J. S. E., Huls, M. H., and Cooper, L. J. N. (2015). 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. Biopharmaceutical Products from Animal Cell Culture, in Animal Cell Culture Cell Engineering. 9, 257267. Trends Biotechnol. Incubate the cells in a humidified 37C, 5% CO. Gently aspirate the media from the cells and wash twice with 250 l 1X PBS. Table 1. As a positive control for transformation, dilute the control pUC19 by 1:5 . trailer <<86899E9D62E54BA7B2F1126FFC054E79>]>> startxref 0 %%EOF 40 0 obj <>stream Electroporation experiments require standard cell culture reagents and instruments appropriate for maintenance of cells. (A) MSCs were electroporated with each one of the seven buffers and the recommended program. 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. 16, 10421049. Whether you are performing your first cloning experiment or constructing multi-fragment DNA assemblies, NEB has the solution for you. (2014). Since multiple loci editing require multiple gRNA, we evaluated the possibility of co-electroporating PBMCs with a reporter plasmid and FITC labeled short RNAs. The .gov means its official. 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). Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments. doi:10.1371/journal.pone.0047868, Gillet, J.-P., Varma, S., and Gottesman, M. M. (2013). Significant differences (p < 0.05) are depicted in the table next to each graph, with each color denoting one parameter. Biomed. In a previous work, our group described seven in house buffers and tested the electroporation efficiency of Jurkat cells and primary lymphocytes using Nucleofector (Chicaybam et al., 2013). Safety and tumor responses with lambrolizumab (AntiPD-1) in melanoma. 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). Learn more by visiting our 4D-Nucleofector X Unit product page or by watching the video. For CRISPR experiments, the plasmid encoding S. pyogenes Cas9 (WT) and a U6 promoter for guide RNA (gRNA) expression was acquired from Addgene (pX330; #42230). doi:10.1371/journal.pone.0060298, de Souza, W. F., Fortunato-Miranda, N., Robbs, B. K., de Araujo, W. M., de-Freitas-Junior, J. C., Bastos, L. G., et al. In this context, the present work comprises a practical guide for the electroporation of 14 cell lines and primary MSCs and HSCs, determining the best buffer (among seven options) to be used with Lonza Nucleofector II, a widely disseminated electroporation device. With an extremely fast plate processing time of one minute and high reproducibility it is the ideal tool for screening applications. 0000005532 00000 n 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). It is a powerful tool for transfecting large DNA fragments and achieving good transfection efficiencies in cell lines. 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). Natl. government site. Cell viability was evaluated after 24 h, and cell expansion was analyzed at day + 1 by crystal violet. For CD34 + cells, around 57% were GFP-positive 1 day after electroporation using buffer 1SM and program U-008 (Figure 6A). 18, 12001209. DMEM with Glutamax (or appropriate growth medium) with 10% FBS Optimized sleeping beauty transposons rapidly generate stable transgenic cell lines. Competitor B electroporation: 25 mV, 96 F. Moreover, this technique is also very efficient, inducing transgene expression levels comparable to viral vectors in some cells (Bilal et al., 2015). Proc. 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. NUCLEOFECTOR is a registered trademark of Lonza Group Ltd. doi:10.1038/sj.gt.3302141, Chicaybam, L., and Bonamino, M. H. (2014). Exp Hematol. 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. An efficient low cost method for gene transfer to T lymphocytes. If you don't see your country above, please visit our GFP-positive colonies (black bars) were determined within total colonies identified. doi:10.1016/j.ijpharm.2011.07.013, Weber, K., Bartsch, U., Stocking, C., and Fehse, B. Representative plots of a high electroporation score cell line (HEL) and a low score cell line (NIH3T3). All the authors read and approved the final manuscript. 0000003955 00000 n Numbers depict the percentages of cells in each gate. doi:10.1073/pnas.2331323100, Park, E. S., Rabinovsky, R., Carey, M., Hennessy, B. T., Agarwal, R., Liu, W., et al. Transgene expression can be enriched by using G418 and is retained after in vivo growth. 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). PLoS ONE 8:e74994. See this image and copyright information in PMC. Find out more by visiting the HT Nucleofector System product pageor watching our video tutorial. Epicentre QuickExtract DNA Extraction Solution (Epicentre #QE09050), 65C for 15 min TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer. The GFP expression can be restored by CRISPR-mediated non-homologous end joining (NHEJ) repair. Rev. This membrane permeability increase is used for many applications in biotechnology, medicine and the food industry. Ther. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Methods Mol. 93, 896908. Cancer Gene Ther. Bioeng. doi:10.1146/annurev-bioeng-071813-104622, Yin, H., Kanasty, R. L., Eltoukhy, A. Molecular evolution of a novel hyperactive sleeping beauty transposase enables robust stable gene transfer in vertebrates. Science 341, 1233151.10.1126/science.1233151 To achieve efficient gene editing of target cells, Cas9 nuclease and the gRNA must be expressed in the cell, ideally in a transient fashion. 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). 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. Seed the cells so that they will be around 70-90% confluent on the day of transfection.
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