Cell transfection is the process of transferring exogenous genetic materials, such as DNA or RNA, into eukaryotic cells to modify the expression profile¹.

Cell transfection has several applications, spanning from studies on gene expression and cellular processes to protein production and gene therapy. It enables researchers to manipulate cells in ways that would otherwise be challenging without altering their genetic makeup. Given its broad range of experimental applications, it is essential for researchers to understand the various types and methods of transfection, ways to quantify its efficiency, potential obstacles, and the most recent advances in the field.

Types of cell transfection

The two main cell transfection techniques are transient and stable transfection^1,3. While both transfection methods serve the common purpose of delivering genetic material into cells, the way this material behaves and is processed within the cellular environment can vary significantly depending on the method used.

Transient transfection

In transient transfection, the foreign genetic material is temporarily introduced into cells. It does not require integrating nucleic acid into the host cell genome. The inserted genes exist in the cytoplasm in the form of a plasmid or as oligonucleotides and usually remain expressed for a short duration (transgene expression is lost with host cell replication). Transient transfection is suitable for high-throughput applications such as gene expression studies (knock-in or knock-down of a particular gene) and when spontaneous, short-term results are required.

Stable transfection

In stable transfection, the foreign genetic material is integrated into the genome of the host cell. Here, cells can continuously express the transfected gene regardless of several rounds of cell division. Stable transfection is a vital tool for any long-term gene expression studies, overexpression of recombinant proteins, or for generating genetically modified cell lines that are aimed at therapy or for future research applications.

Methods of cell transfection

The method of cell transfection depends on different types of cells, experimental needs, and desired outcomes.

Viral-based transfection

The viral-based transfection method, also known as transduction, involves using viral vectors to deliver genetic material into the target cells^1,2,3. This method usually reports higher efficiency, especially in difficult-to-transfect cell types such as primary cells and stem cells. Compared to other methods of transfection, viral vectors offer the advantages of effective gene delivery and can be used for stable and transient transfections.

Viral-based stable transfection:

Stable transfection via viral vectors is typically achieved using retroviruses, including lentiviruses^1,2. These vectors carry an RNA genome that is reverse-transcribed into double-stranded DNA by the viral enzyme reverse transcriptase. This DNA is then integrated into the host cell genome with the help of integrase, resulting in long-term and stable expression of the transgene.

• Retroviruses can efficiently transduce dividing cells but are generally ineffective in non-dividing cells.
• Lentiviruses, a subclass of retroviruses, have the added advantage of being able to transduce both dividing and non-dividing cells, making them more versatile for a wide range of experimental and therapeutic applications⁴.
• Despite their advantages, retroviral integration into the host genome carries the risk of insertional mutagenesis and gene disruption, which may lead to oncogenesis^1,5.

Viral-based transient transfection:

Transient gene expression is typically achieved using non-integrating viral vectors such as adenoviruses, adeno-associated viruses(AAVs), and herpes simplex viruses (HSV). These vectors do not integrate their genetic material into the host genome. Instead, their genomes exist as episomes within the host nucleus, resulting in temporary gene expression that declines over time due to episomal degradation or dilution during cell division.

• Adenoviruses have higher packaging capacities and can transduce a wide variety of dividing and non-dividing cells, but they are associated with higher immunogenicity and cytotoxicity⁶.
• AAVs are considered safer vectors due to their low immunogenicity and pathogenicity⁷. However, their small packaging capacity (<5 kb) limits their use for large gene delivery.
• HSV vectors offer the largest packaging capacity (~150 kb) among common viral vectors and show strong tropism for neuronal cells, making them especially valuable for gene delivery in the nervous system⁶.

Though non-integrating vectors carry a lower risk of genomic disruption, transient expression may not be suitable for long-term transgene expression studies. Additionally, despite being non-integrative, these vectors can still trigger host immune responses and inflammation, particularly in the case of adenoviruses and HSV.

Non-viral-based transfection

Non-viral-based transfection methods are generally preferred because they are simpler and have a lower potential for insertional mutagenesis. However, these methods have lower efficiencies. Below is a brief description of the commonly preferred non-viral-based transfection methods.

Physical methods

The physical methods of non-viral-based transfection use mechanical forces to create temporary pores in the cell membrane of the host cell, thereby allowing the entry of genetic material. These techniques are mainly applied for transfecting cells that are resistant to chemical methods or when precise control over transfection parameters is required.

Electroporation: The electroporation technique uses an electrical field to create temporary pores in the cell membrane, allowing the introduction of genetic material^1,3. It is one of the most common and efficient methods for transfecting mammalian cells. This method is widely used due to its simplicity and effectiveness across various cell types, including primary cells and stem cells.

Transfection efficiencies can vary depending on the cell type and electroporation parameters; for example, fibroblasts may achieve stable transformation frequencies of 1 in 10³ to 10⁴ live cells, while more resistant cell lines may have lower efficiencies. However, the application of high-voltage pulses can lead to cell damage, including necrosis and apoptosis, necessitating careful optimization of electroporation conditions. ​

Gene injection: Gene injection involves direct injection of DNA or RNA into the cells with the help of fine needles or micro-injection systems^1,3. This method is best suited for highly accurate delivery of genetic material, making it ideal for applications such as the creation of transgenic animals and studies involving specific cellular compartments.

However, microinjection is labor-intensive and low-throughput, requiring skilled personnel and specialized equipment. Additionally, the physical manipulation involved can cause damage to cells, necessitating careful technique to maintain cell viability.

Laser-assisted transfection: Laser-assisted transfection, also known as optoporation, employs focused laser beams to create transient pores in the cell membrane, allowing the introduction of genetic material². Techniques such as femtosecond laser pulses can achieve high spatial precision, enabling targeted transfection of single cells with minimal damage.

Other methods, including photochemical internalization, use light-activated compounds to disrupt endosomal membranes, facilitating cytoplasmic release of endocytosed materials³. While optoporation offers precise control, it typically has lower throughput compared to other methods, making it more suitable for applications requiring targeted delivery.

Sonoporation: Ultrasound-assisted transfection, or sonoporation, involves using ultrasonic waves to create transient pores in the cell membrane, facilitating the uptake of nucleic acids such as DNA and RNA. Transfection efficiency is influenced by ultrasound parameters such as pulse duration, exposure time, and intensity up to an optimal threshold, beyond which cell viability declines.

A group of researchers observed higher efficiency when transfecting suspension-cultured 293T cells^1,10. Additionally, it was found that efficiency also varies by cell type; another group found HeLa and T-24 cells responded more favorably to ultrasound than PC-3 or U937 cells^1,11. Thus, optimization based on cell line and culture conditions is essential for successful sonoporation.

Magnetofection: The magnetofection technique uses magnetic fields to direct DNA or nanoparticles into cells that have been preloaded with magnetic particles¹. This technique enhances the concentration of genetic material at the cell surface, promoting uptake through endocytosis^1,3. Magnetofection has been adapted for various vectors, including plasmid DNA and viral particles, and offers advantages such as rapid transfection and reduced cytotoxicity. Though it allows for targeted delivery, it can be less efficient than other methods.

Chemical methods

The chemical methods of non-viral-based transfection use chemical compounds to facilitate the entry of genetic material. These methods are often preferred for their ease of use, scalability, and ability to transfect a wide range of cell types, although their efficiency and cytotoxicity can vary significantly depending on the reagent and experimental conditions.

Microparticles or nanoparticles: These particles are modified to carry genetic material into cells. Their small size enhances cellular uptake. Micro or nanoparticles can also be engineered with surface modifications such as polyethylene glycol (PEG) or ligands to enhance uptake efficiency and biocompatibility. Nanoparticles often consist of materials such as silica, gold, carbon, or biodegradable polymers and can be used alone or in combination with other carriers.

Recent studies suggest that nanoparticles provide a low cytotoxic alternative to traditional methods while enabling high loading capacity and intracellular delivery. However, their efficacy can vary based on particle size, surface charge, and the type of genetic material being delivered. Moreover, the synthesis and characterization of nanoparticles require precise optimization to ensure consistency and reproducibility.

Polymer-based methods: This technique uses biocompatible polymers to encapsulate genetic material through electrostatic complexation to preserve it from degradation and enhance cellular uptake⁸. Cationic polymerssuch as polyethyleneimine (PEI), poly(L-lysine), and chitosan are commonly used. These vectors can provide a controlled release and increase stability, but toxicity can be an issue with some polymers. These polymers not only protect the genetic material from enzymatic degradation but also aid in endosomal escape and facilitate sustained release within cells.

PEI is widely used due to its high transfection efficiency, though it is often associated with significant cytotoxicity, particularly in its high molecular weight and branched forms^1,3. To mitigate this, researchers have developed biodegradable and less toxic alternatives. Controlled release, enhanced stability, and easy surface modification make polymers a versatile choice in gene delivery; however, achieving a balance between efficacy and toxicity remains a challenge.

Peptides/cations: Cationic peptides such as lipids can be used to deliver nucleic acids into the cells⁹. These carriers can merge with the phospholipid bilayer of the cell membrane, promoting uptake via fusion or endocytosis. Like liposomal reagents, cationic lipid-based formulations have shown high transfection efficiencies in a variety of cell types. Although these methods are effective, they are usually toxic at higher concentrations.

Calcium phosphate: This is a traditional method in which the positively charged calcium ions bind with the negatively charged DNA to form precipitates and facilitate DNA transfer into cells by endocytosis and phagocytosis^1,3. It is relatively inexpensive and easy but not compatible with all cell types. Moreover, it requires careful control of conditions such as pH, temperature, and DNA concentration to achieve consistent results.

Dendrimers: Dendrimers are branched macromolecules that facilitate gene delivery into cells¹. They offer a large surface area for binding with genetic material and can be designed to be biocompatible and less toxic.

Although dendrimers are more efficient than calcium phosphate, they still lag behind liposomal and viral systems in terms of transfection efficiency. Hybrid dendrimer systems and surface modifications are being explored to overcome these limitations and enhance performance in clinical applications.

Types of transfected nucleic acids

The choice of nucleic acid depends on the experimental goal, cellular context, and desired duration of expression or silencing, all of which influence the selection of an appropriate transfection method. The primary nucleic acids used in transfection include DNA, RNA, and synthetic oligonucleotides, each offering unique advantages based on their structure and function.

Plasmid DNA (pDNA): This is the most commonly used form of DNA for transfection. It typically exists as a circular, supercoiled molecule and contains essential elements such as a promoter (eg, CMV, EF-1α), an origin of replication, a multiple cloning site, and a selectable marker¹. Supercoiled plasmid DNA usually offers higher transfection efficiency, while linearized DNA facilitates stable integration into the host genome. However, unless integration occurs or selection pressure is applied, plasmid-based expression tends to be transient.

Messenger RNA (mRNA): mRNA is increasingly utilized in applications that require rapid and transient protein expression, such as vaccine development and protein replacement therapies. Unlike plasmid DNA, mRNA does not require nuclear localization for translation, allowing for faster protein synthesis and eliminating the risk of genomic integration. However, its inherent instability and susceptibility to enzymatic degradation present significant challenges. These limitations are commonly addressed through chemical modifications to enhance stability and the use of advanced delivery systems to improve cellular uptake and expression efficiency.

Small RNAs and synthetic oligonucleotides: These include siRNA, miRNA, and shRNA and are widely used for gene knock-down (siRNA and shRNA). siRNA provides highly specific mRNA degradation, while miRNAs regulate gene networks by targeting multiple transcripts¹. Synthetic variants such as agomirs and antagomirs are designed for enhanced stability and functionality. Other oligonucleotides, such as locked nucleic acids (LNAs), offer improved binding affinity and resistance to nuclease activity and can sometimes function without a transfection reagent.

Co-transfection: This involves the simultaneous delivery of multiple nucleic acids, such as a plasmid and a siRNA or CRISPR components, enabling multifaceted experiments like gene editing or pathway studies¹. Although versatile, co-transfection requires careful optimization to balance delivery efficiency and compatibility among different nucleic acid types.

Cell transfection techniques have evolved significantly, allowing for the development of both single-cell and high-throughput transfection approaches that are well-suited for large-scale research, requiring the transfection of thousands of cells in a single step.

Single-cell transfection

Transfection at the level of a single cell is achievable with high-precision instruments, such as micropipette-based microinjection technology¹³. Accurately performed microinjection allows for precise dosing of injected material, which is especially important when transducing more than one foreign material into a single cell. Precise microinjection can accomplish single-cell transfection regardless of the cell type or substance delivered.

Additionally, microfluidic-based single-cell transfections, such as electroporation, mechanoporation, and sonoporation, can efficiently deliver the genetic material into single cells and can easily be applied in vivo. These techniques offer high transfection efficiency and cell viability, making them valuable tools for therapeutic development and diagnostics. For instance, droplet-based microfluidic systems have demonstrated efficient single-cell transfection, particularly in hard-to-transfect suspension cells, by encapsulating individual cells with transfection reagents in microdroplets.

High-throughput transfection

Automation plays a key role in high-throughput transfection, allowing researchers to carry out large-scale experiments efficiently². Electroporation is an easy and rapid method for high-throughput transfection. With optimal electroporation conditions, many cells can be transfected in a short time.

Recent innovations in microfluidic platforms have significantly advanced high-throughput transfection technologies. Microfluidic systems designed for continuous-flow gene delivery offer precise control over key parameters such as flow rate, reagent concentration, and exposure time, thereby enhancing transfection efficiency and reproducibility. Furthermore, integrating micro-arraying techniques with optimized transfection reagents has enabled robust and scalable transfection across a wide range of cell types, supporting basic research and high-throughput screening applications.

Selecting the proper transfection method

Selection of a suitable transfection method depends on various factors, such as cell type, desired outcome, and the efficiency of the method^1,3.

Factors affecting method selection

Cell type: Different cell types exhibit different reactions to the various transfection methods. While lipid-based methods are ideal for adherent cells, electroporation may be the best option for suspension cells. Primary and stem cells are often challenging to transfect; non-liposomal reagents have shown better efficiency in some primary human cells, whereas liposomal reagents like Lipofectamine^® have been more effective in others.

Desired outcomes: It is essential to align the choice of transfection method with the experimental needs. Some methods, such as electroporation and viral transduction, are well-suited for stable expression, while others, such as lipid-mediated transfection, are better for transient expression.

Efficiency: Before beginning the experiment, it is essential to check the transfection efficiencies of different methods. A high transfection efficiency ensures that a larger proportion of cells express the desired gene, leading to more robust and reliable results. The efficiency can vary depending on the method and cell type.

Toxicity: Certain transfection methods can negatively impact cell health, compromising both viability and normal cellular function. Chemical transfection reagents, in particular, may trigger cellular stress responses or even apoptosis. Therefore, selecting transfection strategies with minimal cytotoxicity is essential to preserve the physiological integrity of the target cells. Notably, some studies have shown that non-liposomal reagents tend to exhibit lower toxicity levels compared to their liposomal counterparts in specific experimental settings.

Scalability: Scalability is essential when considering applications that require transfecting large numbers of cells. Methods such as electroporation or viral transduction can be readily scaled up for high-throughput experiments or bio-production purposes.

Recommendations for specific applications

Stem cell transfection for regenerative medicine

Stem cell transfection plays an essential role in regenerative medicine applications due to its ability to differentiate into various cell types. Efficient gene delivery into stem cells is essential for enhancing their therapeutic potential. Viral transfection, particularly using lentiviral vectors, is commonly employed for stable gene delivery into stem cells. Non-viral methods, such as electroporation and the use of nanoparticles, are popularly used for transient gene delivery into stem cells with excellent efficiency and low cytotoxicity.

CHO cell transfection in biopharmaceutical production

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production for recombinant protein expression. Stable transfection methods, such as transposon-based systems, are preferred to establish stable cell lines of CHO with high levels of the desired protein. Transient transfection can also be employed for rapid protein production, especially during early-stage development.

Transfecting primary cells for personalized medicine

Primary cells, which are directly isolated from tissues, are often more difficult to transfect than immortalized cell lines due to their finite lifespan and limited expansion capacity. Electroporation and viral transduction are common choices for transfecting primary cells. Optimization of transfection conditions and the use of cell-specific reagents are essential to achieve satisfactory results while transfecting primary cells.

Measuring transfection efficiency

Measuring the efficiency of the transfection experiments becomes imperative to determine whether the desired genetic material has been successfully introduced into the cells³. This can be achieved through various quantitative and qualitative methods.

Quantitative methods: Involve measuring the expression levels of a reporter gene, such as green fluorescent protein (GFP), or the target protein of interest. These assessments are typically carried out using techniques like flow cytometry, which analyzes fluorescence intensity at the single-cell level; quantitative PCR (qPCR), which detects and quantifies gene expression; or western blot to measure protein expression with high sensitivity and precision.

Flow cytometry: This technique allows for precise quantification of transfection efficiency by measuring the fluorescence intensity of cells expressing a fluorescent protein or harboring a fluorescently labeled nucleic acid³. A novel flow cytometric method has been developed that simultaneously quantifies DNA uptake and protein expression, providing a comprehensive assessment of transfection efficiency. This method has demonstrated reproducibility and standardization, making it suitable for optimizing transfection protocols.

Real-time PCR (qPCR): qPCR quantifies the expression levels of specific nucleic acids introduced during transfection. By measuring the abundance of target sequences, qPCR provides a direct assessment of transfection efficiency, which is particularly useful for analyzing small RNA molecules or plasmids³. ​

Reporter gene assays: Utilizing plasmids that express reporter genes, such as luciferase or β-galactosidase, allows for the indirect measurement of transfection efficiency³. The activity of these reporters correlates with the number of successfully transfected cells, offering a quantitative measure of transfection success. ​

Qualitative methods: These methods include post-transfection visual inspection under fluorescence microscopy or cellular phenotyping.

Fluorescence microscopy: By visualizing cells under a fluorescence microscope, researchers can observe the expression of fluorescent proteins or the presence of fluorescently labeled nucleic acids. This method provides a qualitative assessment of transfection efficiency and can be semi-quantitative when combined with image analysis software. ​

Western blotting and immunofluorescence: These techniques detect and quantify the expression of specific proteins resulting from transfection. Western blotting offers semi-quantitative data on protein levels, while immunofluorescence allows for visualization of protein localization within cells³. Both methods are valuable for assessing the functional outcome of transfection.

Applications of cell transfection

Transfection has widespread applications both in research and biotechnology¹.

Gene expression studies

Cell transfection allows researchers to explore the function of genes by introducing foreign genetic material into cells and observing their effects. It is very effective in functional genomics, especially in studying the roles of individual genes in cellular processes.

Protein production

Cell transfection is a pivotal tool in the production of recombinant proteins. Cells, such as CHO cells, can be transfected with genes encoding therapeutic proteins, which are then harvested and purified for use in drug development or therapeutic applications.

For recombinant protein expression from cell lines: Transfected cells are often used to produce large quantities of recombinant proteins, which can be used for therapeutic purposes.

Co-transfection for multi-protein studies: In some cases, multiple genes are transfected into cells simultaneously, enabling the study of protein-protein interactions and the production of multi-subunit proteins.

Challenges in cell transfection

While cell transfection is an invaluable tool, there are several challenges associated with the technique^1,3,14.

Hard-to-transfect cells

Some cells, such as primary cells, are difficult to transfect. To improve transfection efficiency, these cells often require specialized techniques, such as electroporation or viral vectors.

Balancing between efficiency and cytotoxicity

The majority of transfection techniques, particularly physical methods, are cytotoxic. Hence, creating the right balance between efficient transfection and minimal cell toxicity is vital for successful experiments.

Regulatory and ethical considerations

Transfection in clinical or therapeutic applications should be carried out in accordance with regulatory guidelines. Ethical considerations are imperative, especially in gene editing or transfection of stem cells.

Advances in cell transfection techniques

Recent advancements in transfection techniques have greatly expanded the potential applications of this technology³.

Emerging technologies

High-throughput and in vivo transfection systems and the integration of nanoparticles (lipid nanoparticles) and cell-penetrating peptides point towards endless possibilities in gene delivery.

• High-throughput transfection platforms enable the simultaneous manipulation of thousands of genes across diverse cell types, streamlining large-scale genetic screens and drug discovery processes. These systems are often automated and integrated with imaging and analytical tools for rapid assessment.
• In vivo transfection methods have also progressed, allowing targeted delivery of genetic material directly into tissues and organs, thus advancing gene therapy applications.
• The integration of nanotechnology, such as lipid nanoparticles (LNPs), polymeric nanoparticles, and gold nanocarriers, has further improved delivery efficiency, especially for RNA molecules.
• Similarly, cell-penetrating peptides (CPPs) have shown promise in delivering nucleic acids across cellular membranes with minimal toxicity and immune response.

Innovations in CRISPR-Cas9 delivery

CRISPR-Cas9gene-editing technology has revolutionized the field of genetics. However, the delivery of CRISPR components into target cells remains a significant challenge. Recent innovations have focused on developing non-viral delivery methods, such as lipid nanoparticles and extracellular vesicles, to enhance the safety and efficiency of CRISPR delivery¹⁵. These approaches aim to minimize immunogenicity and improve the precision of gene editing. Advancements in delivery technologies are expanding the potential applications of CRISPR-Cas9 in functional genomics and therapeutic interventions.

Collectively, these advancements in transfection technologies and CRISPR-Cas9 delivery methods are propelling the field of genetic engineering forward, offering new opportunities for research and the development of gene-based therapies.

FAQs

What are the advantages of using viral vectors for cell transfection?

Viral vectors offer several advantages for cell transfection¹⁶. They exhibit high transduction efficiency, ensuring effective gene transfer to target cells. Certain viral vectors, like lentiviral vectors, can integrate their genetic cargo into the host genome, providing sustained therapeutic effects. Viral vectors are also adaptable and can be tailored for specific cell types, tissues, or diseases, offering versatility. They can be used for hard-to-transfect cell types for protein overexpression or knock-down. Some viral vectors can also protect transgenes from biological degradation and efficiently cross cellular barriers.

How does electroporation compare to lipofection for gene delivery?

Electroporationand lipofection are both used for gene delivery, but they differ in method and efficiency^17,18. Electroporation uses electrical pulses to create temporary pores in the cell membrane, whereas lipofection relies on lipid complexes to fuse with the cell membrane. Electroporation works efficiently in human embryonic stem cells and human hematopoietic cells, whereas lipofection can exhibit higher knockout efficiency in HeLa cells. Overall, the choice of transfection technique depends on the cell type, desired outcome, and experimental setup.

What are the challenges of transfecting primary cells versus cell lines?

Transfecting primary cells is often more challenging than working with established cell lines due to their lower transfection efficiency and higher sensitivity to stress¹⁹. Primary cells are less receptive to foreign genetic material, requiring the use of electroporation or viral vectors. Additionally, primary cells have a limited lifespan and more complex physiological conditions, making them harder to maintain. In contrast, cell lines are more robust, easier to transfect, and can be cultured indefinitely, offering more consistent results.

References

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6.      Lee CS, Bishop ES, Zhang R, et al. Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes and diseases. 4(2):43–63 (2017).

7.      Nayerossadat, Maedeh & Ali Nayerossadat N, Maedeh T., Ali P.A. Viral and nonviral delivery systems for gene delivery. Advanced biomedical research. 1(1):27 (2012).

8.      Khan, M. Polymers as efficient non-viral gene delivery vectors: the role of the chemical and physical architecture of macromolecules. Polymers. 16(18), 2629 (2024).

9.      Zhu, L., & Mahato, R. I. Lipid and polymeric carrier-mediated nucleic acid delivery. Expert opinion on drug delivery. 7(10), 1209-1226 (2010).

10.  Zou S, Scarfo K, Nantz MH, et al. Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells. International journal of pharmaceutics. 389(1–2):232–243 (2010).

11.  Feril Jr, L. B., Ogawa, R., Tachibana, K., et al. Optimized ultrasound‐mediated gene transfection in cancer cells. Cancer science. 97(10), 1111–1114 (2006).

12.  Duckert, B., Vinkx, S., Braeken, D., et al. Single-cell transfection technologies for cell therapies and gene editing. Journal of controlled release. 330, 963-975 (2021).

13.  Chow, Y. T., Chen, S., Wang, R., et al. Single cell transfection through precise microinjection with quantitatively controlled injection volumes. Scientific reports. 6(1), 24127 (2016).

14.  Huang, S., Henderson, T. R., Dojo Soeandy, C., et al. An efficient low cost means of biophysical gene transfection in primary cells. Scientific reports. 14(1), 13179 (2024).

15.  Yip B.H. Recent Advances in CRISPR/Cas9 Delivery Strategies. Biomolecules. 10(6), 839 (2020)

16.  Butt, M. H., Zaman, M., Ahmad, A., et al. Appraisal for the potential of viral and nonviral vectors in gene therapy: A review. Genes. 13(8), 1370 (2022).

17.  Tabar, M. S., Hesaraki, M., Esfandiari, F., et al. Evaluating electroporation and lipofectamine approaches for transient and stable transgene expressions in human fibroblasts and embryonic stem cells. Cell journal (Yakhteh). 17(3), 438 (2015).

18.  Mars, T., Strazisar, M., Mis, K., et al. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts. The journal of membrane biology. 248, 273-283 (2015).

19.  Gresch, O., & Altrogge, L. Transfection of difficult-to-transfect primary mammalian cells. Protein expression in mammalian cells: Methods and protocols. 65-74 (2012).