Jamour et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00511-x(2024) 25:15

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Comparing chemical transfection,
electroporation, and lentiviral vectortransduction to achieve optimal transfection
conditions in the Vero cell line
Parisa Jamour1,2, Abbas Jamali3, Arash Ghalyanchi Langeroudi4, Behrouz Ebadi Sharafabad5 and Asghar Abdoli1*
Abstract
Background Transfection is an important analytical method for studying gene expression in the cellularenvironment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma
membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating
the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve ahigh transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques
and introduce a highly efficient method for gene delivery in Vero cells.
Methods In the current study, three transfection methods were used, including chemical transfection,
electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cellline. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based
lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry andfluorescence microscopy to detect GFP-positive cells.
Results Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimaltransfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells.
Conclusion TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency inVero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfectionin the Vero cell line.
Keywords Vero cell line, Chemical transfection reagents, Transfection efficiency, Electroporation, TurboFect, Lentivirus*Correspondence:
Asghar Abdoli
asghar.abdoli7@gmail.com
1
Department of Hepatitis and HIV, Pasteur Institute of Iran, Tehran, Iran2
Student Research Committee, Pasteur Institute of Iran, Tehran, Iran3
Department of Influenza and Other Respiratory Viruses, Pasteur Instituteof Iran, Tehran, Iran4
Department of Microbiology and Immunology, Faculty of Veterinary
Medicine, University of Tehran, Tehran, Iran
5
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,
Tabriz University of Medical Science, Tabriz, Iran
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

Background
Transfection is a widely used laboratory technique thatinvolves the introduction of exogenous nucleic acids
into cells to study gene function and products in a cellular context [1]. The success of transfection depends on
several factors, including the quality of the nucleic acids,
duration of the transfection process, transfection reagent
used, and specific cell lines employed. Localization of
the introduced nucleic acids within the cell is crucial for
determining the efficacy of the procedure, and the degradation of nucleic acids by cellular nucleases such as
deoxyribonuclease II (DNase II) poses a significant challenge [2]. As nucleic acids alone cannot permeate thecell membrane, it is necessary to use suitable carriers
for transportation. To investigate the effect of DNase II
on transfection efficiency, we modulated its expression
levels in transfected cells. Their findings demonstrated
a negative correlation between elevated DNase II levels
and the effectiveness of non-viral DNA delivery vectors,
providing definitive evidence that DNase II constitutes
a significant impediment to successful transfection [3].
Various strategies have been developed for nucleic aciddelivery, including physical, chemical, and viral transduction [4].
The Vero cell line is the most widely used continuous
cell line for the production of viral vectors and vaccines.
Historically, this was the first cell line approved by the
WHO for the production of human vaccines. The Verocell line, derived from the kidney epithelial cells of an
African green monkey, is a continuously passaged cellline that can be propagated for a long period. It is susceptible to various viruses and lacks a responsive cellular pathway that is triggered by interferon expression.
Therefore, it has great potential as a viable option forthe production of viral vaccines. These cells have foundapplications in various research fields such as virology
and toxicology [5]. Therefore, regulatory bodies of theWorld Health Organization have recommended the useof the Vero cell line as the primary continuous cell line
for manufacturing viral vaccines for human use [6].
Chemical transfection strategies involve the use ofvarious substances, such as Ca2 + phosphatepolycationand dendrimers, to facilitate DNA transfer across cell
membranes. Cationic polymers, such as poly-L-lysine,
lipopolyamine, and polyamidoamine, have been studied
as potential vehicles for delivering nucleic acids, showing efficient transfection without damaging the cellular
membranes [7, 8].
Physical transfection methods include microinjection,
optical, biolistic, and electroporation [9]. Electroporation is commonly used method in laboratories to obtainhigher transfection efficiencies. By applying a transienthigh-pressure current pulse, cells undergo a process that
results in the formation of nanometer-level micropores

Page 2 of 12

within their membranes. Consequently, this phenomenon enables the entry of genetic materials, proteins,and other biological molecules into cells. Electroporation offers numerous advantages, owing to its simplicity,
rapidity, reproducibility, safety, and compatibility withvarious cell types. This method is particularly effective
for transecting suspension culture cells, which are typically difficult to transfect. Several factors can influenceboth the electroporation transfection efficiency andpost-transfection cell viability. Therefore, optimizing thetransfection conditions is necessary to ensure the successful transfection of different cellular strains [10].
Virus-mediated transfection or transduction uses viral
vectors to introduce specific nucleic acid sequences into
host cells. Lentiviral transduction is highly efficient fordelivering transgenes to mammalian cells, especially primary cells that are difficult to transfect. However, viraltransduction carries the risk of cytotoxicity and viral
infection. Optimizing transfection conditions is crucial
for achieving high efficiency and consistency, because
different cell strains have specific requirements. The heterogeneity of transfection efficiency among cell types
emphasizes the need for comparative studies to determine the most effective method for each cell type [11].
Enhanced gene transfer techniques specific to Vero cells,
commonly used in vaccine development and gene therapy, are essential for successful gene expression studies.

Materials and methods
Cell culture

The Vero cell line (purchased from the National Cell
Bank of Iran, Tehran) was grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Gibco, Invitrogen, USA) supplemented with 10% heat-inactivated fetal bovine serum(Gibco, Invitrogen, USA) and antibiotics (0.1 mg/mL
streptomycin and 100 U/mL penicillin) (Biosera). Cells
were cultured at 37 °C in a humidified incubator with 5%
CO2.
Plasmid purification

The pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro plasmid
obtained from System Bioscience (CD-513B-1, Australia) was purified using the Miniprep plasmid kit (Qiagen, Germany). The DNA concentration was determinedusing a UV-visible spectrophotometer. Plasmids werestored at -20 °C for subsequent use.
Chemical transfection method
Lipofection

Two commercially available transfection reagents, Lipofectamine™ 2000 (Invitrogen, USA) and TurboFect™
(Invitrogen), were used for transfection. Vero cells wereseeded in 24-well plates at a density of 6 × 104 cells/welland allowed to adhere overnight. To initiate transfection,

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

varying concentrations of the plasmid (pCDH-CMVMCS-EF1-CopGFP-T2A-Puro) ranging from 500 ng to 1
and 2 µg, along with different amounts of the transfection reagents (1–2 and 4 µL), were diluted in 100 µL of
OptiMEM medium and incubated at room temperaturefor 30 min. Subsequently, the mixture was added dropwise to the wells and incubated at 37 °C for 4 h. Following this incubation period, the supernatant was replacedwith a fresh medium containing 10% FBS and 1% antibiotics. After 72 h, flow cytometry was performed to quantify GFP-positive cells. All experiments were performedin triplicates.
X-tremeGENE™ 9 transfection

X-tremeGENE™ 9 DNA transfection reagent (Sigma‒
Aldrich, USA) is a proprietary blend of lipids and otherorganic components supplied in 80% ethanol. This
reagent was used for transfection, following the established protocol. Specifically, 500 ng–1 µg and 2 µg of
plasmid DNA, along with 1–2 and 4 µL of the transfection reagent, were added to the cultured cells. After a
72-h incubation period, the expression level was quantitatively assessed using flow cytometry. All experiments
were performed in triplicates.

Page 3 of 12(pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro) was mixed
with the Vero cell suspension. Ebuffer 2, comprising optimum + HEPES-buffered saline (10 mM, pH∼7.3) andphosphate-buffered sucrose (272 mM, pH∼7.3), was prepared and mixed with the counted Vero cells along with
5 µg of plasmid. Similarly, Ebuffer 3, consisting of RPMI
1640, dipotassium phosphate (10 mM), magnesium chloride (1 mM), and sucrose (250 mM, pH∼7.3), was mixed
with 5 µg of the plasmid. Electroporation was performed
in a 4-mm gapped cuvette and immediately pulsed using
a Gene Pulser Xcell (Bio-Rad, Germany) with the electricparameters set at 850 µF in a square wave. Three different
voltage levels, i.e.,200, 300, and 400 V) were employed.
The resistance was set to 100 Ω and the pulse time was
20 ms. The cells were promptly transferred to a 6-wellplate and cultured in a complete medium containing 12%
FBS and 1% antibiotics. After 72 h of electroporation, thecells were trypsinized, and GFP-positive cells were determined by flow cytometry analysis using the Partec Particle Analysis System. All experiments were performed intriplicates.
Virus-mediated transfection methods
Lentiviral vector production and purification

Commercially available linear polyethylenimines, PEIMAX® (Polysciences, USA) 40 kDa was used as transfection reagent [12]. For transfection each 24-well plate
were seeded with Vero cells at 6 × 104 cells/well. Cells
were incubated at 37 °C in 5% CO2 for 24 h before transfection. Briefly, the amount of 500 ng–1 µg and 2 µg of
plasmid DNA diluted in 50 µL of Opti-MEM was mixed
with 1–2 and 4 µL of PEI MAX® (1 mg/ml stock) andincubated at room temperature for 20 min. Cells wererinsed twice with 1 ml of DMEM supplemented with 1%
GlutaMax (no antibiotics), and the plasmid DNA: PEI
mixture was added to the rinsed cells containing 500 µLof DMEM. After 4 h incubation at 37 C and 5% CO2,transfection medium was replaced with 1 ml of Complete
DMEM medium. transfected cells incubation 48 h andthe GFP-positive cells was determined by flow cytometry.
All experiments were performed in triplicates.

The transfer vector, pCDH-CMV-MCS-EF1-CopGFPT2A-Puro, encodes green fluorescent protein (GFP)
using the cytomegalovirus (CMV) promoter. It also
included puromycin as the selection marker. In the process of viral production, we used an envelope vector
called pMD2G (Addgene,12,259), which expresses the
VSV-G protein, and a packaging vector called psPAX2(Addgene,12,260), which expresses Gag-pol and Tat.
These vectors are based on HIV-1 lentivectors. To generate lentivirus particles, we co-transfected the three plasmids into 5 × 105 HEK 293 Lenti-X cells cultured in 6-wellplates. At 48 and 72 h post-transfection, culture supernatants were collected and filtered using a 0.22 μm filter.
The filtered supernatant was then subjected to ultracentrifugation at 50,000 × g for 4 h to obtain viral pellets.
These pellets were subsequently resuspended in highglucose DMEM, mixed well, and incubated overnight ina shaker. Finally, the viral particles were stored at -80 °C
for future use.

Physical transfection methods
Electroporation

In vitro transduction and determination of the lentiviral
vector titerThree different buffer types and voltage levels wereused for electroporation. Initially, Vero cells were
washed three times with ice-cold phosphate-buffered saline (PBS) and then counted at a concentration of 0.3 ml × 1000,000 cells/ml. The counted cells
were then suspended in Ebuffer 1, which contained
sodium chloride (140 mM) and disodium hydrogen
phosphate (pH∼7.3). Subsequently, 5 µg of plasmid

The transduction process involved serial dilutions ofvector-containing suspensions of 6 × 104 Vero cells that
had been seeded in 24-well plates on the previous day.
Each transduction included 8 µg/mL polybrene (SigmaAldrich) in the transducing inoculum. After 6 h, themedium was changed, and after 3 days, trypsinized cells
were resuspended in PBS. Cell transduction was determined by analyzing GFP-positive cells using Partec flow

PEI MAX® transfection

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

cytometry (Partec, Particle Analysis System). The calculation of transduction titers involved determining thepercentage of cells expressing GFP, with 100,000 cells
used for each individual experiment (n ≥ 3). The virus
titer was calculated using the formula TU/ml = (F × N ×D)/V, where TU/ml represents the amount of functional
viral particles (Transducing Units) in 1 ml of the stock
solution. where F represents the percentage of GFP-positive cells (not exceeding 0.20% for reliable titer estimation), N is the number of cells at the time of transduction,
D is the fold-dilution of the vector used for transduction,and volts is the volume (ml) of the diluted vector samples[13]. All experiments were performed in triplicates.
Cell viability analysis

The viability of each transfection was measured using
trypan blue exclusion and was expressed as a percentageof the initial number of cells.
Flow cytometry assay analysis

The percentage of cells that exhibited fluorescence wasdetermined. (Transfected cells) using a Partec PAS
instrument (Germany). To recognize GFP-positive cells,
untransfected cells (control) were used as GFP-negative controls to establish the gates. Consequently, thegated region was analyzed for green fluorescence (488–508 nm) using a Partec PAS instrument supplied with a
6 W argon laser tuned to 488 nm at an output power of
100 mW. The transfection efficiency was determined by
calculating the percentage of cells showing GFP emission in the gated region. Transfected cells were imaged
using an inverted fluorescence microscope to obtain GFP
fluorescence images. (Olympus BX51, London, UK) witha 10× lens and digital images were captured using a digital camera. The samples were evaluated using FlowJo v10
software (Tree Star Inc., Ashland, OR, USA).
Statistical analysis

All data are shown as mean ± SD. A two-way ANOVA
was performed to compare the means among all groups.
All statistical analyses were performed using GraphPad
Prism version 9.0. Differences were considered to be statistically significant at p ≤ 0.05.

Results
Optimizing the transfection efficiency of chemical-based
reagents in the Vero cell line

To determine the highest transfection efficiency bychemical transfection reagent in the Vero cell line, 0.5, 1,
and 2 µg of purified pCDH plasmid with 1, 2, and 4 µL of
chemical transfection reagents, including Lipofectamine™
2000, TurboFect™, X-tremeGENE™ 9, and PEI MAX®,
were transfected into Vero cells.

Page 4 of 12

The highest transfection efficiency was obtained with
1 µg plasmid to 2 µL Lipofectamine™ 2000 (Fig. 1A), and
GFP transfection efficiency at this ratio was significantly
different from the other ratios (p ≤ 0.05). As shown in
Fig. 1A, there was a significant difference in other ratiosof plasmid to Lipofectamine™ 2000, but the maximum
transfection efficiency was observed at this ratio, with
41% GFP-positive cells. As shown in Fig. 2A, cell viability (∼93%) in this cell line using Lipofectamine™ 2000
was comparable, regardless of the ratio. No significant
differences in cell viability were observed between control cells, which did not receive any DNA, and cells transfected with different reagent/DNA ratios (p ≤ 0.05).
The results in Fig. 1B show that TurboFect™ had thehighest transfection efficiency at various DNA concentrations compared to the other transfection reagents. The
highest transfection efficiency was achieved with a DNA/
reagent ratio of 1 µg: 4 µL, which was significantly different from the other ratios. The presence of 46.5% GFPpositive cells with this DNA/reagent ratio confirmedthat the concentrations of both reagents and DNA weresuitable for the Vero cell line. Thus, TurboFect™ showed
the best transfection efficiency compared with the othertransfection reagents used in this experiment. As shownin Fig. 2B, the cell viability (∼94%) of this cell line using
TurboFect™ was comparable, regardless of the ratio. No
significant differences in cell viability were observed
between control cells and cells transfected with different
reagent/DNA ratios post-transfection (p ≤ 0.05, Fig. 2B).
According to the results obtained from Fig. 1c,
X-tremeGENE™ 9 transfection reagent showed optimumtransfection efficiency at a rate of 1 µg:4 µL of DNA/
reagent. The estimated optimum number of GFP-positive
cells was 24.5%, which was significantly different fromthe other DNA/reagent ratios (p ≤ 0.05, Fig. 1C). Cell viability (∼91%) was observed at this ratio, and there wereno significant differences between these and the control
cells (p ≤ 0.05, Fig. 2C).
The results obtained from the PEI MAX® transfection
reagent showed high transfection efficiency in 1 µg:2
µL of DNA/reagent ratio, and GFP-positive cells were
16.5% (Fig. 1D). There was a significant difference compared with the DNA/reagent ratio (p ≤ 0.05). Cell viability (∼89%) was observed at this ratio and there were no
significant differences between the control cells (p ≤ 0.05,
Fig. 2D). A comparison of cell viability results in different
transfection buffers showed that the percentage of Verocell viability after transfection with the PEI MAX® reagent
was lower than that of the control and other transfection
buffers. This suggests that PEI MAX® may be more toxic
to Vero cells than other buffers.

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

Page 5 of 12

Fig. 1 GFP transfection efficiency analysis chemical-based transfection reagents. Vero cell lines were subjected to transfection using Lipofectamine™
2000 (A), TurboFect™ (B), X-tremeGENE™ 9 (C), and PEI MAX® (D). (p ≤ 0.05 *, p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****)

Optimizing the electroporation conditions for different
buffers and voltages in the Vero cell line

To demonstrate better electroporation conditions in the
Vero cell line, three buffers were examined at three voltages. Figure 3A shows the results of flow cytometry of
GFP-positive cells at voltages of 200, 300, and 400 v andEbuffer1, 2, and 3 with various compositions and controls. The maximum transfection efficiency was observedin Ebuffer3 at 300 V (∼38.8%) (Fig. 3B), and the increase
was statistically significant compared to that in Ebuffer1
at the same voltage (p ≤ 0.01). Therefore, the observed cell
viability (∼67%) was compared to that of the control at
this voltage (Fig. 3C). As shown in Fig. 3B, the increasein transfection efficiency at 300 V between buffers 1
and 3 was statistically significant. This increase may be
attributed to the buffer composition, especially RPMI
1640, which was used for successful electroporation in
Vero cells. As shown by the flow cytometry results, thetransfection efficiency at 200 V was lower than that at
300 V, possibly because of insufficient voltage to open
the plasma membrane pores and transfer DNA into thecell. Furthermore, analysis of cell viability data and flow
cytometry results at 400 V showed that cell death was

higher at this voltage than at other voltages, which may
explain the lower percentage of GFP-positive cells andtransfection efficiency.
Optimizing lentiviral vector pseudotype production andtransduction in Vero cell lines

Lentivirus particles were produced in the HEK 293 LentiX cell line by co-transfection with three HIV-1-based
lentivectors. The supernatant of the cells was collected
at 48 and 72 h after transfection. Figure 4A shows GFP
expression in cells at 24, 28, and 72 h after transfection.
As shown in the images, more viral particles formed overtime, which caused morphological changes in cells andthe appearance of cytopathic effects of the virus on cells.
Viral particles were ultracentrifuged for purification. The
viruses were then transduced into Vero cells at dilutions(D) of 1, 4, 16, and 64, and the efficiency of transduction
was measured by flow cytometry to detect GFP expression in the cells. Figure 4B shows the flow cytometry
results for the different viral dilutions transduced into
Vero cells. The highest transduction efficiency (∼15.2%)
was observed at a dilution of one virus. The titer of the
produced lentiviruses was calculated to be 1.9 × 105 (TU/

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

Page 6 of 12

Fig. 2 Cell viability analysis using chemical transfection reagents. Vero cell line viability was assessed using the chemical transfection reagents Lipofectamine™ 2000 (A), TurboFect™ (B), X-tremeGENE™ 9 (C), and PEI MAX® (D)ml) using the formula described above and the percentage of GFP expression in transduced cells. Figure 4C
shows a graph of GFP expression levels in Vero cells
transduced at different dilutions, which shows that theincrease in expression at a dilution of 1 was statisticallysignificant compared to other viral dilutions. It is possible that the low transduction efficiency of lentiviruses in
Vero cells compared to other cells is due to the inhibitory
activity of TRIM5 protein isoforms in the cell.
Comparison of transfection methods for achieving thehighest transfection efficiency in Vero cells

Different transfection methods for assessing optimaltransfection in the Vero cell line were used. A comparison of transfection methods based on chemical reagentsthat showed the highest transfection efficiency revealed
that transfection with TurboFect™ resulted in a statistically significant increase in transfection efficiency compared to other methods (p ≤ 0.05), and PEI MAX® had alower transfection efficiency rate (Fig. 5A). As shownin Fig. 4A, other chemical transfection reagents also
showed statistically significant differences in transfection
efficiency; however, Turbofect had the highest efficiency.
Figure 5B shows a comparison between all transfection

methods, including lentivirus transduction, electroporation, and TurboFect™ (which is a better chemical transfection method). The results revealed that the TurboFect™
transfection reagent method had the highest transfection
efficiency compared with other methods. These results
suggest that TurboFect™ has high potential for efficient
DNA transfer to Vero cells.

Discussion
Molecular biology research often involves the introduction of nucleic acids into eukaryotic cells through transfection, which requires appropriate methods depending
on cell type and research objectives. Each method hasvarying levels of transfection efficiency and cell toxicity[14]. However, the ideal method should exhibit a hightransfection efficiency with minimal toxicity. Optimization is often necessary to determine optimal transfection
conditions [15]. The Vero cell line plays a crucial role invaccine research; however, an optimized transfection
protocol for this cell type is currently lacking. Several
factors must be addressed to achieve optimal transfection, including intracellular uptake, endosome acidification, autophagy, immune sensing pathways, and nuclear
entry [16]. Numerous factors can influence transfection

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

Page 7 of 12

Fig. 3 GFP transfection efficiency analysis of electroporation by flow cytometry demonstrated the expression of GFP at various voltages and Ebuffer in
Vero cells by flow cytometry (A), and analysis showed that there was a significant difference between Ebuffer 3 and Ebuffer 1 at 300 V (B) (p ≤ 0.01**).
Comparison of cell viability in all groups with the control showed that the cell death rate was higher at 400 V than at the other voltages (C)outcomes. Our focus was to introduce an optimal transfection method for the Vero cell line, which involved careful selection and fine-tuning of the transfection reagent
as well as the development of an optimized protocol. In

addition, factors such as cell density, incubation time,
and the reagent-to-DNA ratio should be considered. Systematically evaluating and adjusting these variables cansignificantly improve the transfection efficiency in VeroJamour et al. BMC Molecular and Cell Biology(2024) 25:15

Page 8 of 12

Fig. 4 Lentiviral vector phenotype transduction in Vero cells. GFP expression in HEK293 Lenti-X cells at 24, 48, and 72 h post-transduction (A). Flow cytometry analysis of 1,4,16,64 virus dilution (D) transduced into Vero cells (B) and transfection efficiency in all dilutions in (C)cells, resulting in more reliable and reproducible experimental results [17].
The success of non-viral DNA delivery methods relies
on various factors, including the ability of DNA to evadelysosomal degradation, efficient nuclear translocation, decreased cytotoxicity, better control of molecularcomposition, flexibility in gene size, and lower immunogenicity compared to viral analogs [18]. Studies haveshown that lysosomal degradation can be overcome
through strategies such as the proton sponge effect incertain cationic polymers, such as PEI. However, efficient
nuclear targeting remains a significant challenge during

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

Page 9 of 12

Fig. 5 Comparison of all transfection methods. Comparison of transfection with chemical reagents showed that TurboFect had the highest transfection
efficiency among other reagents in Vero cells (A). A comparison of all transfection methods showed that the use of chemical reagents, especially TurboFect, is still the most successful method for transferring DNA to Vero cells (B). (p ≤ 0.05 *, p ≤ 0.01**, p ≤ 0.001***, p ≤ 0.0001****)

the transfection process. Transfection outcomes can besignificantly influenced by factors, such as cell membranecomposition and pH, as highlighted by Kim and Eberwine [5].
In this study, we investigated the optimal transfection
conditions for the Vero cell line using various chemical transfection agents, including Lipofectamine™ 2000,
TurboFect™, X-tremeGENE™ 9, and PEI MAX®. We
tested different ratios of DNA and chemical transfection
reagents to determine the most effective combination.
Our results showed that using 1 µg of each plasmid with
4 µL of TurboFect™ in 6 × 104 cells achieved the highest
transfection efficiency (46.5%) and cell viability (94%)
compared to other reagents, such as Lipofectamine™
2000 (42% transfection efficiency and 94% cell viability).
By determining the optimal ratio of reagent to DNA,
researchers can enhance the success of transfection
experiments while minimizing any negative effects oncell viability. TurboFect™ is a cationic polymer that forms
complexes with nucleic acids, thereby facilitating cellattachment, internalization, and endosomal escape. It
is particularly suitable for transecting primary cells, difficult-to-transfect cells, and other cell types [19]. Lipofectamine is a cation-lipid-based transfection reagent
that forms liposomes to deliver nucleic acids to cells. The
composition and structure of liposomes can affect theefficiency and effectiveness of transfection experiments[20].
TurboFect™ has a lower toxicity than Lipofectamine,
which contributes to its higher transfection efficiency andcell viability. Choosing TurboFect™ allowed researchers toachieve a balance between effective transfection and highcell viability in Vero cells. In a similar study, CHO-K1

and SH-SY5Y cell lines were transfected with chemical transfection reagents including Lipofectamine 2000,
TurboFect 8.0, and ExGen 500. The results showed thattransfection efficiency with TurboFect reagent was significantly higher in SH-SY5Y cells compared to the othertwo types of chemical transfection reagents, and wasapproximately the same in CHO-K1 cells. The results ofthis study are consistent with the results of our study andmay indicate the success of TurboFect in optimal DNA
delivery in other cell lines [21]. We observed a lower
transfection efficiency with X-tremeGENE™ 9 and PEIMAX®. PEI, a cationic polymer, exhibited higher cytotoxicity. It can induce toxicity by depolarizing mitochondria
and stimulating immune responses. High-molecularweight PEI (HMW PEI) can form stable polyplexes, but
its non-cleavable structure increases its cytotoxicity [22].
PEI– DNA complexes can activate genes involved in cellular responses including apoptosis, stress responses, andoncogenesis [23].
To reduce the cytotoxicity of PEI, researchers can use
degradable disulfide-containing polymers, which increase
the disassembly rate of the complexes and reduce theirbinding affinity with intracellular membranes [24]. In
addition, free cationic PEI chains embedded in the cell
membrane can enhance transfection efficiency by destabilizing the endosomal membrane and hindering its
fusion with lysosomes [25].
Overall, our study highlights the importance of selecting an appropriate transfection reagent and the ratio ofreagent to DNA to optimize transfection efficiency andmaintain cell viability in Vero cells.
Electroporation transfection is a physical method used
to introduce external substances into the cells. It involves

Jamour et al. BMC Molecular and Cell Biology(2024) 25:15

subjecting cell membranes to high-voltage pulses, which
creates tiny pores that allow for the insertion of exogenous genes [26]. Several factors influence the transfection
efficiency and cell viability during electroporation. These
factors include the electric field strength (voltage), pulseinterval (capacitance flow rate), temperature, buffer composition, cell state and volume, and the concentrationand structure of foreign genes [27]. Among these factors,
electric field strength (voltage) and buffer composition
are particularly important in determining transfection
efficiency.
In this study, we aimed to identify the optimal electroporation conditions by testing three different electricfield intensities (200, 300, and 400 V) and three different
buffers with varying ingredients on Vero cell lines. The
results showed that all three buffers exhibited the highest transfection efficiency at 300 V, with an electric pulseinterval of 850 µF in square waves at all voltages. The
cells demonstrated varying levels of adaptation to bothelectric field intensity and pulse interval. If the voltage
is below optimal levels or the electroporation conditions
are inadequate for a specific cell strain, no changes occur
in the cell membranes, hindering the entry of exogenous
genes and reducing the transfection efficiency [28]. Conversely, excessive voltage leads to irreversible cell damage, which significantly affects both cell survival andtransfection efficiency [29]. In a similar study investigating the optimal conditions for electroporation of skeletal
muscle satellite cells, it was demonstrated that increasing
voltage intensity resulted in a decrease in electroporation
efficiency and cell viability. DNA dosage was also foundto have a significant impact on electroporation success.
The optimal voltage at which the highest electroporation
efficiency and cell viability were observed varied depending on the cell type, highlighting the importance of determining the optimal electroporation conditions for eachcell type [30].
In addition, the results demonstrated that Ebuffer 3
exhibited higher transfection efficiency (38.6%) thanthe other buffers, and this increase was statistically significant. The composition of the buffer significantly
influenced the transfection efficiency. Using RPMI-1640
medium as a shock buffer simplifies this process andreduces cell damage and death after transfection. RPMI1640 without serum and antibiotics showed higher cell
survival after shock than other buffers [10]. Transfection
and viability remained unaffected when RPMI-1640 wasused as the transfection buffer, even for cells cultured indifferent media [31].
However, under optimal transfection conditions, thecell viability after electroporation was 56%, which wassignificantly lower than that of the control. One notable
limitation of the electroporation transfection technique
is its inherent cellular toxicity, which can range from 50

Page 10 of 12

to 90%. In general, electroporation maintains viability
within the range of 30–40% and can be further optimized
to maximize transfection efficiency.
HIV-1 lentiviral vectors are commonly used for efficient gene delivery and long-term genetic modificationin various cell types, including dividing and non-dividing
cells [32]. These vectors offer high titers and low risk ofgenerating replication-competent retroviruses, making them a safe and powerful tool for gene transfer [33].
However, our research indicates that lentivirus particles
have a low transduction efficiency in Vero cells, even athigh titers, with a reported maximum efficiency of 20%.
These findings align with those of previous studies, suggesting that the low transduction efficiency in Vero cells
may be due to post-entry restrictions against HIV-1. In
particular, innate cellular responses in Vero cells can
impede the uncrating process of the virion capsid structure, primarily through the activity of TRIM5 protein
isoforms, which possess a defense mechanism against
the virus known as ubiquitin ligase activity [34]. Basedon these findings, HIV-1 lentiviral vectors are not suitable for efficient transduction of Vero cells. Therefore,
alternative lentiviruses derived from different viruses oralternative transfection methods should be considered toachieve optimal and efficient gene transfer into Vero cells[35].
Additionally, we compared various transfection methods to identify the optimal conditions for transfectionin Vero cells. The results demonstrated that TurboFect™,
a chemical transfection reagent, exhibited the highest
transfection efficiency in Vero cells. Non-viral delivery
methods, such as chemical transfection, offer advantages
over viral vector delivery, including reduced immunogenicity and a lower risk of insertional mutagenesis [36].
Statistically, TurboFect™ significantly outperformed otherchemical transfection methods such as electroporationand transduction using lentiviral vectors. Electroporation, although effective for gene delivery, resulted in ahigh rate of cell death, which negatively affected transfection efficiency. In contrast, transduction using lentiviral
vector-based HIV-1 showed low efficiency in Vero cells
because of the presence of an intracellular inhibitor for
HIV-1 integration [37]. It can be presumed that the highefficiency of TurboFect™ in creating optimal conditions
in Vero cells is attributed to the formation of liposome
particles, their successful transfer through the lipid membrane, evasion from degradation by lysosomes, and efficient nuclear translocation.

Conclusions
Efficient transfection of DNA into cells is a valuable
tool in molecular biology for applications such as genetherapy, recombinant virus production, and vaccine
generation. Owing to their unique characteristics, VeroJamour et al. BMC Molecular and Cell Biology(2024) 25:15

cells have been used in many studies, including vaccination and virology. Therefore, it would be very valuable tointroduce a transfection method with the highest DNA
transfer efficiency. Our research findings demonstrate atransfection efficiency of 46.5% when using 1 µg of DNA
with 4 µL of TurboFect™ in 6 × 104 cells, which is the highest transfection rate in this cell line. Other transfection
methods, such as electroporation, did not yield goodresults because of high cell mortality. In addition, transduction of lentivectors based on HIV-1 virus inhibits replication of HIV-1 lentivectors in the Vero cell line.
Abbreviations
DNase II	deoxyribonuclease II
DMEM	Dulbecco’s modified Eagle’s medium
CMV	cytomegalovirus
GFP	green fluorescent protein
TU/ml	Transducing units in 1 ml
PEI	Polyethylenimine
HIV-1	human immunodeficiency virus-1
Acknowledgements
The authors would like to thank the personnel at the Pasteur Institute of Iran.
Author contributions
Conceptualization: A.A, P.J, A.J- Funding acquisition: A.A- Investigation: P.J, A.A,
B.E- Supervision: A.A, A.G, A.J- Writing–review & editing: P.J, A.J, A.G, B.E, A.A
and all authors read and approved the final manuscript.

Page 11 of 12

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Funding
This study was funded as a PhD student project by the Pasteur Institute of Iran,
Tehran (Grant Number: bd-9787).

19.

Data availability
All data generated or analysed during this study are included in this publishedarticle.

21.

Declarations

20.

22.

Ethics approval and consent to participate
Not applicable.

23.

Consent for publication
Not applicable.

24.

Competing interests
The authors declare that they have no conflicts of interest.
Received: 24 January 2024 / Accepted: 17 April 2024

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