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Abstract

Background:

Models of muscle cell culture allow simple manipulation to optimize gene transfer experiments, offering a significant opportunity to model muscle effects by allowing precise control of the cell culture environment in a cost-effective manner. Often, gene transfer is performed in myoblasts, which are then differentiated into myotubes, a skeletal muscle model. In this study, we aimed to compare the efficiency of direct transfection of differentiated myotubes by chemical and physical plasmid methods.

Methods:

Differentiated myotubes were transfected with reporter plasmids. Transfection efficiency and cell death were quantified. Proinflammatory proteins are produced by cells and tissues after DNA transfection; therefore, the secretion of proinflammatory chemokines was quantified.

Results:

In this study, we demonstrate that C2C12 myotubes can be effectively transfected using multiple methods. While a chemical method did not cause cell death, myotubes were not efficiently transfected. Electroporation delivery produced a higher transfection efficiency coupled with higher cell death. A subset of proinflammatory chemokine proteins was secreted, which replicated secretion by skeletal muscle.

Discussion:

These results suggest that differentiated myotubes can be directly transfected invitro, which may represent a useful experimental model. However, more studies are needed to assess the value of this model in predicting invivo responses.

Introduction

The three Rs in animal welfare are replacement, reduction, and refinement.1 When developing gene therapies, replacement with cells or models avoids the use of animals to the fullest extent possible. Undifferentiated cells in culture are often studied; however, differentiated cells may be a more appropriate model for tissues. Although there is no replacement for muscle invivo, the simple differentiation of myoblasts to myotubes can mimic aspects of skeletal muscle tissue invivo.2 –4 In this study, experiments were performed in the C2C12 myotube model, an environment potentially more relevant to invivo studies than the myoblast. This model was used to compare nonviral gene transfer methods.

Several technologies produce efficient delivery of plasmid deoxyribonucleic acid (pDNA) transfection into cells and tissues, including chemical and physical methods.5 We compared direct transfection of C2C12 myotubes using two common methods, several chemical reagents, and electroporation (electrotransfer). This study first aimed to assess transfection efficiency and myotube survival.

Intramuscular injection of plasmid DNA is inflammatory.6 Intramuscular DNA electrotransfer is associated with the production of proinflammatory molecules7,8 and inflammation.9 –11 These inflammatory effects may be due to the activation of pattern recognition receptor pathways, which are ubiquitously expressed in mammalian cells. In cells with functional signaling pathways, endosomal or cytosolic DNA sensors are activated by the introduction of exogenous nucleic acids inherent in DNA-based therapies.12 Therefore, a second aim was to evaluate the expression of proinflammatory chemokines induced by DNA transfection of myotubes to determine whether this expression was similar to that detected after invivo skeletal muscle delivery.

Materials and Methods

Cells and myotube formation

C2C12 mouse myoblast cells (CRL-1772, American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (Corning, Thermo Fisher Scientific, USA) supplemented with 5% fetal bovine serum (Gibco, Waltham, MA, USA) and 1x Penicillin-Streptomycin (Gibco) in a 5% CO₂ humidified incubator at 37°C. The cells regularly tested negative for mycoplasma infection using the Myco-Sniff Polymerase Chain Reaction Detection Kit (MP Biochemicals, Irvine, CA, USA). C2C12 myoblasts at 60-80% confluency were differentiated into myotubes by starvation for 8 days in medium containing 2% horse serum without antibiotics as previously described.13

pDNA transfection

Plasmids encoding Katushka fluorescent protein (pTurboFP635-C, Evrogen, Moscow, Russia) or luciferase (gWiz Luc, Aldevron, Fargo, ND, USA) driven by the cytomegalovirus-immediate early promoter/enhancer were commercially prepared (Aldevron, Fargo, ND, USA). Endotoxin levels were confirmed to be <100 endotoxin units/mg. Myoblasts and differentiated myotubes were transfected using several proprietary reagents, Transit LT1 (Mirus Bio LC, Madison, WI, USA), EndoFectin Max (GeneCopoeia, Rockville, MD, USA), and jetPrime (PolyPlus Transfection, New York, NY, USA) per the manufacturer's instructions. For electrotransfer, differentiated C2C12 myotubes in complete medium containing a final concentration of 0.25 µg/µL pDNA were electroporated using a BTX ECM 830 Square Wave Electroporator coupled with a Petri-Pulser electrode (PP35-2P, Harvard Apparatus, Holliston, MA, USA). A single 10 ms 160 V square wave pulse was delivered.14 Conditioned medium was collected after 48 h incubation in complete culture medium.

Luciferase Quantification

Four hours after transfection, culture medium was replaced with medium containing 150 µg/mL d-luciferin (Gold Biotechnology, St. Louis, MO, USA). Luciferase activity in relative light units was immediately quantified (CLARIOStar, BMG Labtech, Cary, NC, USA).

Microscopic analysis

Crystal violet staining was performed as previously described15 and visualized microscopically. The number of myotubes was quantified using ImageJ.16 For Live–Dead staining, the medium was replaced 48 h after electrotransfer with two drops each of room temperature NucBlue^® Live reagent and NucGreen^® Dead reagent (ReadyProbes Cell Viability Imaging Kit, Thermo Fisher Scientific, Waltham, MA, USA) to 1 mL of medium.

Fluorescence was monitored microscopically (BZ-X700E, Keyence Corp., Itasca, IL, USA). Live cells were detected with a standard DAPI (4′,6-diamidino-2- phenylindole) blue filter set (excitation/emission maxima: 360/460 nm), and the nuclei of dead cells with compromised plasma membranes were detected with a standard fluorescein isothiocyanate/green fluorescent protein (green) filter set (excitation/emission maxima: 504/523 nm). A standard ET-mCherry, Texas Red^® (Chroma Technology Corporation, Bellows Falls, VT), filter set (excitation/emission maxima: 560/630) was used to detect Katushka fluorescent protein expression. Fluorescence was quantified using the Hybrid Cell Count tool (Keyence). After loading the files in .jpg format into the software, we manually delimited the myotubes to specify the extraction area. The myotubes, defined as containing three or more live (NucBlue stained) nuclei, were tracked and counted using Image J software.12 Using the tool, the number of blue/live cells (NucBlue) and green/dead cells (NucGreen) within the delimited areas was then counted. For transfection efficiency, the red (Katushka expressing myotubes) were quantified and normalized to total viable myotubes. Assessors were blinded to the experimental groups until the completion of the study.

Chemokine quantification by bead array

Conditioned medium from electroporated myotubes was analyzed using a premixed multiplex panel (Mouse Cytokine/Chemokine Magnetic Luminex Assay, Millipore, Burlington, MA, USA) on a MAGPIX System (Luminex, Austin, TX, USA) per the manufacturer's instructions. All protein identifiers are per UniProt.17

Statistical analysis

Statistical evaluation of the differences between groups and graph preparation was conducted using GraphPad Prism 9.1.0 (San Diego, CA, USA). Since the data were normally distributed, significance was determined by a one-way ANOVA test followed by a Tukey-Kramer post-test. A p < 0.05 was considered statistically significant.

Results

Myoblast differentiation

Several culture conditions can be used to induce myotube formation from myoblasts.4 As observed previously, starvation of C2C12 myoblasts in 2% horse serum produced multinucleated myotubes13,15 (Fig.1).

FIG. 1.

Evaluation of C2C12 myotube differentiation. Representative brightfield images of crystal violet-stained cells. Myotube images were taken in sequence during differentiation. Images are shown on (a) day 0 and (b) day 8. Scale bar = 200 μm. (c) Quantification of 27 myotubes per group (****p < 0.0001).

pDNA delivery via chemical methods

Myoblast transfection was quantified after pDNA delivery using three commercial proprietary reagents (Fig.2). While each reagent significantly delivered plasmid DNA as denoted by luciferase transgene expression (p < 0.0001), jetPrime produced significantly higher expression than the other reagents or the plasmid only control (p < 0.0001).

FIG. 2.

Luciferase expression 24 h after gWiz Luc delivery using proprietary reagents. C, control; E, Endofectin Max; T, Transit LT1, J, jetPrime. ****p < 0.0001 compared to control. n = 4 per group.

We next transfected differentiated C2C12 myotubes in situ with jetPrime. After 48 h, we examined the degree of transfection by detecting Katushka expression by fluorescence microscopy (Figs.3a and S1a). Cell death was not associated with transfection (Figs.3b and S1b). No significant transgene expression was observed in the naïve, pDNA only, and reagent only groups; the transfection efficiency in the DNA electrotransfer group was approximately 8% (Figs.3c and S1c).

FIG. 3.

Live and dead staining of differentiated myotubes transfected with pTurboFP635-C (Katushka) via jetPrime. (a) Fluorescence microscopy representative images of dead cells (green), live cells (blue), and transfected cells (red). Scale bar = 200 µm. (b) Quantification of the number of myotubes containing live (blue), dead (green), and red (transfected). (c) Quantification of transfected myotubes. n = 3–6 fields of view per group. ***p < 0.001, *p < 0.05.

pDNA delivery via electrotransfer

To examine whether C2C12 myotubes exhibited efficient pDNA transfection by electrotransfer, we electroporated differentiated C2C12 myotubes with pDNA (Figs.4a and S2a). Viability, as indicated by the number of myotubes containing blue nuclei, decreased by 35% (p < 0.0001) in the myotubes exposed to pDNA electrotransfer when compared to control myotubes (Figs.4b and S2b). No significant transgene expression was observed in the naïve, pDNA only, and pulse only groups; the transfection efficiency was approximately 36% in the group receiving pDNA electrotransfer (Figs.4c and S2c).

FIG. 4.

Live and dead staining of differentiated myotubes transfected with pTurboFP635-C (Katushka) via electroporation (EP). (a) Fluorescence microscopy representative images of dead cells (green), live cells (blue), and transfected cells (red). Scale bar = 200 µm. (b) Quantification of the number of myotubes containing live (blue), dead (green), and red (transfected). (c) Quantification of transfected myotubes. n = 3–6 fields of view per group. ***p < 0.001, *p < 0.05.

Chemokine expression

We quantified a subset of chemokine proteins secreted into the medium by differentiated myotubes 48 h after pDNA electrotransfer (Fig.5). After 8 days of differentiation, the myotubes were treated with pDNA and/or pulses. Protein expression in supernatants was evaluated using a multiplex bead array. Pulse application didnot induce chemokine expression. CXCL2 was downregulated after the introduction of pDNA. However, C-C motif chemokine ligand 3 (CCL3), CCL4, CCL5, and C-X-C motif chemokine ligand 10 (CXCL10) were significantly secreted after pDNA electrotransfer.

FIG. 5.

Cytokines and chemokines detected in mouse myotube medium 48 h after plasmid electroporation (EP). CCL, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand. ***p < 0.001, **p < 0.01, *p < 0.05 compared to control. n = 4–5 per group.

Discussion

Differentiation of murine C2C12 myoblasts is frequently used as a myotube model to mimic the invivo differentiation process invitro. C2C12 myotubes recreate many aspects of the development,2 contraction,3 and biomechanical properties4 of skeletal muscle myotubes, although direct comparisons are rare. With starvation, the majority of C2C12 cells pause in G1 after growth in differentiation medium, leave the cell cycle, and eventually differentiate and fuze to form multinucleated myotubes.13

Different proprietary formulations are complexed with DNA to enhance transfection. Transit LT1 (Mirus Bio) contains an amphipathic compound and a DNA-binding protein,18 EndoFectin Max (GeneCopoeia) is a lipid-based formulation, while jetPrime (PolyPlus Transfection) is a cationic polymer-based reagent. Each of these formulations successfully transfected C2C12 myotubes. Although high cell viability was maintained, the transfection efficiency was only 8%. Differentiated cells do not divide, and it is well-accepted that breakdown of the nuclear envelope during mitosis is optimal for nuclear entry of gene therapy vectors, particularly nonviral vectors.19,20 This may explain the level of transfection efficiency. Myotubes may transfect more easily during process associated with differentiation such as cell–cell membrane fusion. However, this study was designed to mimic fully differentiated rather than developing or regenerating muscle.

Myoblast plasmid transfection followed by differentiation into myotubes has been reported using electroporation,3,21,22 chemical methods,3,22 –24 or viral methods.25 Sandri etal. reported the transfection of 3-day-old myotubes seeded on coverslips by electrotransfer.26 In this study, five 20 ms pulses at an applied voltage to distance ratio of 180 V/cmat 200 ms intervals were delivered using spatula-like electrodes. Luciferase expression and green fluorescence protein immunofluorescence staining were used to confirm transfection. Transfection efficiency was not measured. While our study varied in the pulse protocol, electrode, plasmid, and DNA concentration, our data confirmed the successful transfection of differentiated myotubes using pDNA electrotransfer. Myoblast transfection efficiency was previously published using two different pulse protocols and is approximately 20% as measure by flow cytometry.7 Interestingly, this efficiency may be lower than the observed myotube transfection level of 36%.

Plasmid DNA can be repeatedly electroporated intomuscles and cells due to its low immunogenicity,27 although acute inflammatory responses7,8 and inflammation9 –11 have been reported. We observed the production of proinflammatory chemokines by myotubes. The expression pattern of myotubes did not necessarily mirror those of skeletal muscle 4 h after pDNA electrotransfer.8 Both myotubes and skeletal muscle secreted the chemokines CCL3, CCL4, and CXCL10 in response to pDNA electrotransfer. However, while myotubes secreted CCL5, skeletal muscle did not. Overall, skeletal muscle was much more responsive to pDNA exposure. Skeletal muscle produced increased CXCL2 in response to either pDNA injection or pDNA electrotransfer, while CXCL2 production was downregulated in myotubes with pDNA exposure. These differences may be due to the presence of other cell types invivo, the assay time point or the concept that tissue injection is different from simple DNA exposure in culture. Clearly, invitro systems may not entirely mimic tissue responses. The production of inflammatory chemokines generally supports the hypothesis that these responses may be due to the detection of plasmid DNA by endosomal or cytosolic DNA sensors such as cyclic guanosine monophosphate–adenosine monophosphate synthase and other putative sensors initiating inflammatory signaling.28,29 A multiplex bead array was used to detect expression of a therapeutic protein, IL-12p70, as well as its downstream effector interferon-γ, after intratumor electrotransfer.30

Finally, myotube stress may be induced by pulse application. By definition, pulse application induces permeabilized areas, commonly known as “pores,” in the cell membrane.31,32 Transcriptional changes in addition to inflammatory pathways are induced.33 The production of reactive oxygen species during pulse application has been described.34,35 It is well-established in several cell types that cells undergo transient cytoskeletal reorganization after pulse application.36 –44

Conclusions

In many applications, myotubes differentiated invitro from myoblasts can be a potential model for skeletal muscle. Here, we demonstrate that myotubes can be successfully transfected in situ by electroporation. However, the biological responses of myotubes, as indicated by chemokine expression in this study, may not entirely parallel those of skeletal muscle. This may limit the translational importance of observations in this model.

Author Contributions: ASC: conceptualization, methodology, validation, formal analysis, investigation, writing – original draft, writing – review & editing. AC: investigation, formal analysis, writing – review & editing. JGA: investigation, formal analysis, writing – review & editing. MB: investigation, formal analysis, writing – review & editing. SP: investigation, formal analysis, writing – review & editing. JS: methodology, formal analysis, investigation, writing – review & editing. RH: resources, writing – review & editing. LCH: conceptualization, methodology, validation, formal analysis, writing – review & editing, funding acquisition.

Conflicts of Interest: The authors declare that they have no conflicts of interest.

Funding Statement: This research was supported in part by the National Cancer Institute of the National Institutes of Health [award number R01CA196796] and by the Department of Medical Engineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

Russell

WMS

, Brunch

RL.

The Principles of Humane Experimental Technique. London, UK: Universities Federation for Animal Welfare, 1959.

Burattini

, Ferri

, Battistelli

, et al. C2C12 murine myoblasts as a model of skeletal muscle development: Morpho-functional characterization. Eur J Histochem. 2004; 48(3): 223–233.

Manabe

, Miyatake

, Takagi

, et al. Characterization of an acute muscle contraction model using cultured C2C12 myotubes. PLoS One. 2012; 7(12): e52592. DOI: 10.1371/journal.pone.0052592.

Ren

, Song

, Liu

, et al. Molecular and biomechanical adaptations to mechanical stretch in cultured myotubes. Front Physiol. 2021; 12(689492). DOI: 10.3389/fphys.2021.689492.

Fus-Kujawa

, Prus

, Bajdak-Rusinek

, et al. An overview of methods and tools for transfection of eukaryotic cells invitro. Front Bioeng Biotechnol. 2021; 9(701031). DOI: 10.3389/fbioe.2021.701031.

McMahon

, Wells

, Bamfo

, et al. Inflammatory responses following direct injection of plasmid DNA into skeletal muscle. Gene Ther. 1998; 5(9): 1283–1290.

Semenova

, Bosnjak

, Markelc

, et al. Multiple cytosolic DNA sensors bind plasmid DNA after transfection. Nucleic Acids Res. 2019; 47(19): 10235–10246. DOI: 10.1093/nar/gkz768.

Sales Conniff

, Tur

, Kohena

, et al. Transcriptomic analysis of the acute skeletal muscle effects after intramuscular DNA electroporation reveals inflammatory signaling. Vaccines (Basel). 2022; 10(12). DOI: 10.3390/vaccines1012:2037.

Babiuk

, Baca-Estrada

, Foldvari

, et al. Electroporation improves the efficacy of DNA vaccines in large animals. Vaccine. 2002; 20(27–28): 3399–3408. DOI: 10.1016/s0264-410x(02)00269-4.

10.

Babiuk

, Pontarollo

, Babiuk

, et al. Induction of immune responses by DNA vaccines in large animals. Vaccine. 2003; 21(7–8): 649–658. DOI: 10.1016/s0264-410x(02)00574-1.

11.

Babiuk

, Baca-Estrada

, Foldvari

, et al. Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines. J Biotechnol. 2004; 110(1): 1–10.

12.

Paludan

, Bowie

AG.

Immune sensing of DNA. Immunity. 2013; 38(5): 870–880. DOI: 10.1016/j.immuni.2013.05.004.

13.

Soltow

, Zeanah

, Lira

, et al. Cessation of cyclic stretch induces atrophy of C2C12 myotubes. Biochem Biophys Res Commun. 2013; 434(2): 316–321. DOI: 10.1016/j.bbrc.2013.03.048.

14.

Geiger

, Taylor

, Glucksberg

, et al. Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer. Gene Ther. 2006; 13(8): 725–731. DOI: 10.1038/sj.gt.3302693.

15.

Contreras

, Villarreal

, Brandan

Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation. Skelet Muscle. 2018; 8(1): 5. DOI: 10.1186/s13395-018-0150-5.

16.

Rueden

, Schindelin

, Hiner

, et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinform. 2017; 18(1): 529. DOI: 10.1186/s12859-017-1934-z.

17.

UniProt

UniProt: The universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49(D1): D480–D489. DOI: 10.1093/nar/gkaa1100.

18.

Wolff

, Fritz

, Budker

, et al. Process of Transfecting a Cell with a Polynucleotide Mixed with an Amphipathic Compound and a DNA-Binding Protein. Madison, WI: Mirus Corporation, 1998.

19.

Tseng

, Haselton

, Giorgio

TD.

Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochim Biophys Acta. 1999; 1445(1): 53–64. DOI: 10.1016/s0167-4781(99)00039-1.

20.

Brunner

, Sauer

, Carotta

, et al. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther. 2000; 7(5): 401–407. DOI: 10.1038/sj.gt.3301102.

21.

Espinos

, Liu

, Bader

, et al. Efficient non-viral DNA-mediated gene transfer to human primary myoblasts using electroporation. Neuromuscul Disord. 2001; 11(4): 341–349. DOI: 10.1016/s0960-8966(00)00204-2.

22.

Miyake

, Hayashi

, Iwasaki

, et al. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-beta in myoblasts. Biochem Biophys Res Commun. 2010; 393(4): 762–766. DOI: 10.1016/j.bbrc.2010.02.077.

23.

Liu

, Wang

, Zhao

, et al. MyoD-dependent induction during myoblast differentiation of p204, a protein also inducible by interferon. Mol Cell Biol. 2000; 20(18): 7024–7036. DOI: 10.1128/mcb.20.18.7024-7036.2000.

24.

Sabourin

, Harisseh

, Harnois

, et al. Dystrophin/alpha1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes. Cell Calcium. 2012; 52(6): 445–456. DOI: 10.1016/j.ceca.2012.08.003.

25.

Jackson

, Hoversten

, Powers

, et al. Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: Comparing and optimizing approaches. FEBS J. 2013; 280(3): 827–839. DOI: 10.1111/febs.12072.

26.

Sandri

, Bortoloso

, Nori

, et al. Electrotransfer in differentiated myotubes: A novel, efficient procedure for functional gene transfer. Exp Cell Res. 2003; 286(1): 87–95. DOI: 10.1016/s0014-4827(03)00097-1.

27.

Sokolowska

, Blachnio-Zabielska

AU.

A critical review of electroporation as a plasmid delivery system in mouse skeletal muscle. Int J Mol Sci. 2019; 20(11): 2276. DOI: 10.3390/ijms20112776.

28.

Motwani

, Pesiridis

, Fitzgerald

KA.

DNA sensing by the cGAS-STING pathway in health and disease. Nat Rev Genet. 2019; 20(11): 657–674. DOI: 10.1038/s41576-019-0151-1.

29.

Unterholzner

The interferon response to intracellular DNA: Why so many receptors?. Immunobiology. 2013; 218(11): 1312–1321. DOI: 10.1016/j.imbio.2013.07.007.

30.

Burkart

, Mukhopadhyay

, Shirley

, et al. Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters. Gene Ther. 2018; 25(2): 93–103. DOI: 10.1038/s41434-018-0006-y.

31.

Weaver

JC.

Electroporation: A general phenomenon for manipulating cells and tissues. J Cell Biochem. 1993; 51(4): 426–435. DOI: 10.1002/jcb.2400510407.

32.

Neumann

, Kakorin

, Toensing

Fundamentals of electroporative delivery of drugs and genes. BioelectrochemBioenerg. 1999; 48(1): 3–16. DOI: 10.1016/s0302-4598(99)00008-2.

33.

Sales Conniff

, Tur

, Kohena

, et al. DNA electrotransfer regulates molecular functions in skeletal muscle. Bioelectricity. epub. DOI: 10.1089/bioe.2022.0041.

34.

Bonnafous

, Vernhes

, Teissie

, et al. The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane. Biochim Biophys Acta. 1999; 1461(1): 123–134. DOI: 10.1016/s0005-2736(99)00154-6.

35.

Pakhomova

, Khorokhorina

, Bowman

, et al. Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media. Arch Biochem Biophys. 2012; 527(1): 55-64. DOI: 10.1016/j.abb.2012.08.004.

36.

Kanthou

, Kranjc

, Sersa

, et al. The endothelial cytoskeleton as a target of electroporation-based therapies. Mol Cancer Ther. 2006; 5(12): 3145–3152. DOI: 10.1158/1535-7163.MCT-06-0410.

37.

Hall

, Schoenbach

, Beebe

SJ.

Nanosecond pulsed electric fields have differential effects on cells in the S-phase. DNA Cell Biol. 2007; 26(3): 160–171. DOI: 10.1089/dna.2006.0514.

38.

Stacey

, Fox

, Buescher

, et al. Nanosecond pulsed electric field induced cytoskeleton, nuclear membrane and telomere damage adversely impact cell survival. Bioelectrochemistry. 2011; 82(2): 131–134. DOI: 10.1016/j.bioelechem.2011.06.002.

39.

Yizraeli

, Weihs

Time-dependent micromechanical responses ofbreast cancer cells and adjacent fibroblasts to electric treatment. CellBiochem Biophys. 2011; 61(3): 605-618. DOI: 10.1007/s12013-011-9244-y.

40.

Rosazza

, Escoffre

, Zumbusch

, et al. The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells. Mol Ther. 2011; 19(5): 913–921. DOI: 10.1038/mt.2010.303.

41.

Meulenberg

, Todorovic

, Cemazar

Differential cellular effects of electroporation and electrochemotherapy in monolayers of human microvascular endothelial cells. PLoS One. 2012; 7(12): e52713. DOI: 10.1371/journal.pone.0052713.

42.

Mao

, Wang

, Chang

, et al. Involvement of a Rac1-dependent macropinocytosis pathway in plasmid DNA delivery by electrotransfection. Mol Ther. 2017; 25(3): 803–815. DOI: 10.1016/j.ymthe.2016.12.009.

43.

Graybill

, Davalos

RV.

Cytoskeletal disruption after electroporation and its significance to pulsed electric field therapies. Cancers. 2020; 12(5). DOI: 10.3390/cancers1205:1132.

44.

Bhandary

, Sales Conniff

, Miranda

, et al. Acute effects of intratumor DNA electrotransfer. Pharmaceutics. 2022; 14(10). DOI: 10.3390/pharmaceutics1410:2097.

Direct Plasmid DNA Transfection into Differentiated Mouse C2C12 Myotubes

Abstract

Background:

Methods:

Results:

Discussion:

Introduction

Materials and Methods

Cells and myotube formation

pDNA transfection

Luciferase Quantification

Microscopic analysis

Chemokine quantification by bead array

Statistical analysis

Results

Myoblast differentiation

pDNA delivery via chemical methods

pDNA delivery via electrotransfer

Chemokine expression

Discussion

Conclusions

References