AccScience Publishing / GPD / Volume 2 / Issue 2 / DOI: 10.36922/gpd.0353
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Optimization of transfection methods for human lymphoblast TK6 cell line

Akamu Jude Ewunkem1* Kyle Agee1
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1 Department of Biological Science, Winston Salem State University, Winston Salem, North Carolina, USA
Submitted: 21 March 2023 | Accepted: 5 May 2023 | Published: 29 May 2023
© 2023 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( )

Transfection has recently gained attention in the field of biomedical research due to its ability to manipulate gene expression. Every mammalian cell type has a characteristic set of requirements for optimal transfection. Some cells can be difficult to transfect and require optimization for successful transfection. Human lymphoblast TK6 cell line, an important cell line for genotoxic studies, is known to be extremely hard to transfect. Thus, optimizing transfection methods for human lymphoblast TK6 are increasingly important. To accomplish this, TK6 human lymphoblasts were transfected with plasmid constructs that expressed green fluorescent protein (GFP) and NanoLuc® activity. We compared the transfection efficiency of three commercially available transfection reagents, including Amaxa 96-well Nucleofection procedure using various solutions (SF, SE, and SG), Lipofectamine LTX, and Metafectene Pro®. The transfection efficiency and toxicity of various reagents were tested by fluorescence microscopy, luciferase activity, and cell viability assays. Amaxa 96-well Nucleofection Solution SF was identified as the best transfection reagent due to its relatively high luciferase activity, acceptable cell viability (80%), and GFP transfection efficiency (80%). Optimal conditions for transfection utilized with this reagent included 0.4 μg of plasmid DNA, 1.8 × 105 cells, and using the DS 137 Nucleofector program.

Lipofectamine LTX
Metafectene Pro
Cell viability
National Institute of Environmental Health Sciences AREA
National Institute of General Medical Sciences MBRS SCORE
  1. Zu Y, Huang S, Lu Y, et al., 2016, Size specific transfection to mammalian cells by micropillar array electroporation. Sci Rep, 6(1): 38661–38610.


  1. Alabdullah AA, Al-Abdulaziz B, Alsalem H, et al., 2019, Estimating transfection efficiency in differentiated and undifferentiated neural cells. BMC Res Notes, 12(1): 225.


  1. Lieberman JR, Ghivizzani SC, Evans CH, 2002, Gene transfer approaches to the healing of bone and cartilage. Mol Ther, 6(2): 141–147.


  1. Avci-Adali M, Behring A, Keller T, et al., 2014, Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression. J Biol Eng, 8(1): 8.


  1. Praveen BB, Stevenson DJ, Antkowiak M, et al., 2011, Enhancement and optimization of plasmid expression in femtosecond optical transfection. J Biophotonics, 4(4): 229–235.


  1. Cerda MB, Batalla M, Anton M, et al., 2015, Enhancement of nucleic acid delivery to hard-to-transfect human colorectal cancer cells by magnetofection at laminin coated substrates and promotion of the endosomal/lysosomal escape. RSC Adv, 5(72): 58345–58454.


  1. Keller AA, Scheiding B, Breitling R, et al., 2019, Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania. J Cell Biochem, 120(1): 14–27.


  1. Ensenauer R, Hartl D, Vockley J, et al., 2011, Efficient and gentle siRNA delivery by magnetofection. Biotech Histochem, 86(4): 226–231.


  1. Kroeger K, Collins M, Ugozzoli L, 2009, The preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation. J Vis Exp, 6(23): 1026.


  1. McCoy AM, Collins ML, Ugozzoli LA, 2010, Using the gene pulser MXcell electroporation system to transfect primary cells with high efficiency. J Vis Exp, 35: 1662.


  1. Sridharan A, Patel C, Muthuswamy J, 2013, Voltage preconditioning allows modulated gene expression in neurons using PEI-complexed siRNA. Mol Ther Nucleic Acids, 2: e82.


  1. Nyamay’Antu A, Dumont M, Kedinger V, et al., 2019, Non-viral vector mediated gene delivery: The outsider to watch out for in gene therapy. Cell Gene Ther Insights, 5: 51–57.


  1. Thomas CE, Ehrhardt A, Kay MA, 2003, Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet, 4(5): 346–358.


  1. Karra D, Dahm R, 2010, Transfection techniques for neuronal cells. J Neurosci, 30(18): 6171–6177.


  1. Thumann G, Stöcker M, Maltusch C, et al., 2010, High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor. Gene Ther, 17(2): 181–189.


  1. Mehier-Humbert S, Guy RH, 2005, Physical methods for gene transfer: Improving the kinetics of gene delivery into cells. Adv Drug Deliv Rev, 57(5): 733–753.


  1. Schenborn ET, Goiffon V, 2000, DEAE-dextran transfection of mammalian cultured cells. Methods Mol Biol, 130: 147–153.


  1. Smolders S, Kessels S, Smolders SM, et al., 2018, Magnetofection is superior to other chemical transfection methods in a microglial cell line. J Neurosci Method, 293: 169–173.


  1. Park JS, Surendran S, Kamendulis LM, et al., 2011, Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies. BMC Res Notes, 4(1): 8.


  1. Alatorre-Meda M, González-Pérez A, Rodríguez JR, 2010, DNA-METAFECTENE™ PRO complexation: A physical chemistry study. Phys Chem Chem Phys, 12(27): 7464–7472.


  1. Maucksch C, Aneja MK, Hennen E, et al., 2008, Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase. Nucleic Acids Res, 36(17): 5462–5471.


  1. Riccetti L, Sperduti S, Lazzaretti C, et al., 2019, Glycosylation pattern and in vitro bioactivity of reference follitropin alfa and biosimilars. Front Endocrinol (Lausanne), 10: 503.


  1. Hoser SM, Hoffmann A, Meindl A, et al., 2020, Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing. Genome Biol, 21(1): 299.


  1. Kataoka H, Saeki A, Hasebe A, et al., 2021, Naringenin suppresses Toll‐like receptor 2‐mediated inflammatory responses through inhibition of receptor clustering on lipid rafts. Food Sci Nutr, 9(2): 963–972.


  1. Kim DY, Park JS, Leem YH, et al., 2021, The potent PDE10A inhibitor MP-10 (PF-2545920) suppresses microglial activation in LPS-induced neuroinflammation and MPTP-induced parkinson’s disease mouse models. J Neuroimmune Pharmacol, 16(2): 470–482.


  1. Vozza-Brown L, Fan J, Vasu S, et al., 2015, Lipofectamine™ LTX: A New Transfection Reagent for Effective Transfection of Primary Cells, Hard-To-Transfect Cells and Sensitive Established Cell Lines. San Francisco: Invite Corp.


  1. Ishiguro K, Watanabe O, Nakamura M, et al., 2017, Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts. Biotechniques, 63(1): 37–39.


  1. Dalby B, Cates S, Harris A, et al., 2004, Advanced transfection with Lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods, 33(2): 95–103.


  1. Nematollahi MH, Torkzadeh-Mahanai M, Pardakhty A, et al., 2018, Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: A comparative study with protamine and lipofectamine. Artif Cells Nanomed Biotechnol, 46(8): 1781–1791.


  1. Wang C, Du M, Huang D, et al., 2019, Inhibition of PARP1 increases IRF-dependent gene transcription in Jurkat cells. Curr Med Sci, 39(3): 356–362.


  1. Duarte FB, Brigido MD, de Oliveira Melo ED, et al., 2020, Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector. Anim Biotechnol, 33: 1014–1024.


  1. Taghavi M, Parham A, Dehghani H, et al., 2021, Optimizing lipofectamine LTX complex and G-418 concentration for improvement of transfection efficiency in human mesenchymal stem cells. Arch Razi Inst, 76: 1315–1325.


  1. Nakayama A, Sato M, Shinohara M, et al., 2007, Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system™. Cloning Stem Cells, 9(4): 523–534.


  1. Dityateva G, Hammond M, Thiel C, et al., 2003, Rapid and efficient electroporation-based gene transfer into primary dissociated neurons. J Neurosci Methods, 130(1): 65–73.


  1. Lenz P, Bacot SM, Frazier-Jessen MR, et al., 2003, Nucleoporation of dendritic cells: Efficient gene transfer by electroporation into human monocyte-derived dendritic cells. FEBS Lett, 538(1–3): 149–154. https://doi.org10.1016/ s0014-5793(03)00169-8


  1. Ganesh L, Burstein E, Guha-Niyogi A, et al., 2003, The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes. Nature, 426: 853–857.


  1. Schakowski F, Buttgereit P, Mazur M, et al., 2004, Novel non-viral method for transfection of primary leukemia cells and cell lines. Genet Vaccines Ther, 2(1): 1.


  1. Leclere PG, Panjwani A, Docherty R, et al., 2005, Effective gene delivery to adult neurons by a modified form of electroporation. J Neurosci Methods, 142(1): 137–143.


  1. Yin J, Ma Z, Selliah N, et al., 2006, Effective gene suppression using small interfering RNA in hard-to-transfect human T cells. J Immunol Methods, 312(1–2): 1–11. https://doi. org10.1016/j.jim.2006.01.023


  1. Hu B, Zou Y, Zhang L, et al., 2019, Nucleofection with plasmid DNA for CRISPR/Cas9-mediated inactivation of programmed cell death protein 1 in CD133-specific CAR T cells. Hum Gene Ther, 30(4): 446–458.


  1. Duan C, Hu D, Tang X, et al., 2019, Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites. Mol Biochem Parasitol, 228: 1–5.


  1. Herrero AB, Quwaider D, Corchete LA, et al., 2020, FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma. J Cell Mol Med, 24(7): 4171–4182.


  1. Ihle M, Biber S, Schroeder IS, et al., 2021, Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices. Nucleic Acids Res, 49(13): 7457–7475.


  1. Hasan MM, Ragnarsson L, Cardoso FC, et al., 2021, Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain. PLoS One, 16(3): e0243645.


  1. O’mahoney JV, Adams TE, 1994, Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol, 13(12): 1227–1232.


  1. Mancinelli S, Turcato A, Kisslinger A, et al., 2021, Design of transfections: Implementation of design of experiments for cell transfection fine‐tuning. Biotechnol Bioeng, 118: 4488–4502.


  1. Aluigi MG, Hofreiter S, Falugi C, et al., 2007, Efficiency of two different transfection reagents for use with human NTERA2 cells. Eur J Histochem, 51(4): 301–304.


  1. Greene E, Cazacu D, Tamot N, et al., 2021, Optimization of a transient antibody expression platform towards high titer and efficiency. Biotechnol J, 16(4): 2000251.


  1. Timin AS, Muslimov AR, Lepik KV, et al., 2018, Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid micro-carriers. Nanomedicine, 14(1): 97–108.


  1. Attali-Padael Y, Armon L, Urbach A, 2021, Apoptosis induction by the stem cell factor LIN28. Biol Cell, 113: 450–457.


  1. Harris E, Zimmerman D, Warga E, et al., 2021, Nonviral gene delivery to T cells with Lipofectamine LTX. Biotechnol Bioeng, 118(4): 1674–1687.


  1. Reilly NM, Pittman DL, 2019, A mammalian genetic complementation assay for assessing cellular resistance to genotoxic compounds. In: DNA Repair. New York: Humana, p209–215.


  1. Auld DS, Inglese J, 2018, Interferences with luciferase reporter enzymes. In: Assay Guidance Manual. United States: Bethesda.


  1. Parson CD 2nd, 2015, A Generalizable Method to Normalize Luciferase Activity in Transiently Transfected B cells (Doctoral Dissertation, North Carolina Agricultural and Technical State University).


  1. Orth P, Weimer A, Kaul G, et al., 2008, Analysis of novel non-viral gene transfer systems for gene delivery to cells of the musculoskeletal system. Mol Biotechnol, 38(2): 137–144.


  1. Ziemann C, Rüdell G, Riecke A, 2002, METAFECTENE Application Note. Available from: con_4_6_4/cms/upload/pdf/ziemanne.pdf [Last accessed on 2023 Mar 04].


  1. Mollah MM, Kim Y, 2021, HMGB1‐like dorsal switch protein 1 of the mealworm, Tenebrio molitor, acts as a damage‐associated molecular pattern. Arch Insect Biochem Physiol, 107: e21795.


  1. Kim Y, Ahmed S, Al Baki MA, et al., 2020, Deletion mutant of PGE2 receptor using CRISPR-Cas9 exhibits larval immunosuppression and adult infertility in a Lepidopteran insect, Spodoptera exigua. Dev Comp Immunol, 111: 103743.


  1. Hunt MA, Currie MJ, Robinson BA, et al., 2010, Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents. J Biomol Tech, 21(2): 66–72.


  1. Shi B, Xue M, Wang Y, et al., 2018, An improved method for increasing the efficiency of gene transfection and transduction. Int J Physiol Pathophysiol Pharmacol, 10(2): 95–104.


  1. Cheung WY, Hovey O, Gobin JM, et al., 2018, Efficient nonviral transfection of human bone marrow mesenchymal stromal cells shown using placental growth factor overexpression. Stem Cells Int, 2018: 1310904.


  1. Yoshinaga N, Uchida S, Naito M, et al., 2019, Induced packaging of mRNA into polyplex micelles by regulated hybridization with a small number of cholesteryl RNA oligonucleotides directed enhanced in vivo transfection. Biomaterials, 197: 255–267.


  1. Ma Y, Sekiya M, Kainoh K, et al., 2020, Transcriptional co-repressor CtBP2 orchestrates epithelial-mesenchymal transition through a novel transcriptional holocomplex with OCT1. Biochem Biophys Res Commun, 523(2): 354–360.


  1. Hock DH, Reljic B, Ang CS, et al., 2020, HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV. Mol Cell Proteomics, 19(7): 1145–1160.


  1. Ewert KK, Ahmad A, Bouxsein NF, et al., 2008, Non-viral gene delivery with cationic liposome-DNA complexes. In: Gene Therapy Protocols. United States: Humana Press, p159–175.


  1. Vijayanathan V, Thomas T, Thomas TJ, 2002, DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry, 41(48): 14085–14094.


  1. Hughes MM, Hooftman A, Angiari S, et al., 2019, Glutathione transferase omega-1 regulates NLRP3 inflammasome activation through NEK7 deglutathionylation. Cell Rep, 29(1): 151–161.e5.


  1. Schiøtz BL, Rosado EG, Baekkevold ES, et al., 2011, Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection. BMC Res Notes, 4(1): 136.


  1. Marrone L, Bus C, Schöndorf D, et al., 2018, Generation of iPSCs carrying a common LRRK2 risk allele for in vitro modeling of idiopathic Parkinson’s disease. PLoS One, 13(3): e0192497.


  1. Nimmo IC, Barbrook AC, Lassadi I, et al., 2019, Genetic transformation of the dinoflagellate chloroplast. Elife, 8: e45292.


  1. Song HY, Chien CS, Yarmishyn AA, et al., 2019, Generation of GLA-knockout human embryonic stem cell lines to model autophagic dysfunction and exosome secretion in fabry disease-associated hypertrophic cardiomyopathy. Cells, 8(4): 327.


  1. Badakov R, Jaźwińska A. Efficient transfection of primary zebrafish fibroblasts by nucleofection. Cytotechnology, 51(2): 105–110.


  1. Sandbichler AM, Aschberger T, Pelster B, 2013, A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line. Biores Open Access, 2(1): 20–27.


  1. Chicaybam L, Sodre AL, Curzio BA, et al., 2013. An efficient low cost method for gene transfer to T lymphocytes. PLoS One, 8(3): e60298.


  1. Satkauskas S, Bureau MF, Puc M, et al., 2002, Mechanisms of in vivo DNA electrotransfer: Respective contributions of cell electropermeabilization and DNA electrophoresis. Mol Ther, 5(2): 133–1340.


  1. Rothmann-Cosic, K, Wessendorf, H, Helfrich, J, et al., 2002, Buffer solution for electroporation and a method comprising the use of the same. Europäische Patentschrift EP, 1390518, B1.
Conflict of interest
There are no conflicts of interest to declare.
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Gene & Protein in Disease, Electronic ISSN: 2811-003X Published by AccScience Publishing