An Introduction to Transfection, Transfection Protocol and Applications

The ability to alter the genetic composition of living cells has revolutionized biology. Scientific advances, from treating genetic disorders through gene therapy to reprograming skin cells into neurons, have been made possible by the increasing proficiency in introducing foreign nucleic acids (DNA and RNA) into cells using a technique known as transfection.

In 1928, Griffith proposed the “transforming principle” having observed that bacterial cells could take up foreign hereditary genetic material.¹This led to the discovery of DNA as that genetic material,²a discovery that enabled great strides in science. In the 1960s, viruses were employed to transfer genetic material into animal cells in a controlled manner and it was shown that foreign genetic material could be expressed in animal cells; this opened up the possibility of gene therapy.³The simultaneous advances in the field of recombinant technology – the discovery of plasmids⁴followed by an increased application of plasmids in the 1970s and the discovery of restriction enzymes^5,6– facilitated the manipulation of genes. Around the same time, chemical and physical methods of introducing the genetic material into cells, such aselectroporation,⁷calcium phosphate transfection⁸and liposomal⁹transfection were also developed, thus providing a plethora of methods to deliver the modified genes into cells of interest.

With the development of transfection methods, many discoveries in basic and translational sciences have been possible and the technique has a plethora of applications in biology. This includes understanding the role of target genes in healthy and diseased cells, unraveling molecular pathways, designing gene therapeutic approaches, cellular reprograming and many more. Thus, transfection is now an indispensable molecular and cell biology laboratory technique. In this article, we discuss the fundamentals of transfection and provide an overview of some of the commonly employed methods. A sample protocol that can be used as a starting point is included and finally, we consider some of the key applications of transfection.

Transfection is a commonly used technique employed to transfer foreign nucleic acids intoeukaryotic cells.¹⁰The purpose of transfection is to alter the genetic content of the host cells, thus changing the expression of desired genes in these cells.

It is important here to distinguish between the terms transfection and transformation. While the term transfection is used when the host cells are eukaryotic, the term transformation is used to denote the transfer of nucleic acids to bacterial cells. This distinction is vital because in higher eukaryotic cells, transformation refers to the process by which the cells become malignant.¹¹

The primary objective of a transfection technique is to ensure that the desired foreign nucleic acid can cross the cell membrane and that a substantial amount of that nucleic acid is protected from degradation to allow its expression within the cell. The transfer of nucleic acids into host cells can be achieved through various physical, chemical and biological methods. In most of these cases, the cellular uptake of the nucleic acids is mediated throughendocytosis¹²of the nucleic acid along with a carrier (Figure 1). A portion of these nucleic acids can avoid lysosomal degradation through what is known asendosomal escapeand make their way to the nucleus where they can be transcribed to affect gene expression. While this is not an exhaustive list of the methodologies employed to achieve transfection, we summarize here some of the most commonly used transfection methods.

Figure 1:广义mec的图解表示hanism of transfection.

Physical methods including electroporation and gene gun

Physical methods of transfection apply electrical, mechanical or thermal forces¹³to facilitate nucleic acid entry into host cells. Some examples includemicroinjection,electroporation,biolistic transfection(gene gun),sonoporation,magnetofectionandlaser optoporation. While the microinjection method employs a special needle to inject the nucleic acids directly into the cells, the other physical methods involve inducing transient and reversible permeabilization of the cell membrane while simultaneously placing the nucleic acids in the vicinity of the permeabilized membrane.¹⁴In electroporation, short and intense electrical pulses are applied to achieve transient permeabilization of the cell membrane. Similarly, ultrasound waves achieve transient cell membrane permeabilization in the case of sonoporation; and the same effect is achieved using controlled exposure to a laser beam in the case of laser optoporation. Biolistic approaches propel naked DNA coated with heavy metal particles into the cell using gas discharge. Magnetofection utilizes magnetic nanoparticles to guide the nucleic acids to the cell membrane where they can be taken up by the process of endocytosis. Physical methods of transfection have the advantage that they do not pose an immunogenic risk like viral methods and are not restricted in the length of the nucleic acid sequences that can be used like viral and some chemical methods. However, these methods require dedicated and expensive equipment and reagents, and they often offer low transfection efficiencies with high cellular mortality.

Chemical methods including lipofection

A number of chemical reagents have been developed to assist DNA/RNA to cross the cell membrane.¹⁵These includecationic lipids,calcium phosphate,cationic polymersandnanoparticles. Transfection using cationic liposomal lipids is termed aslipofection和涉及的形成带正电的lipid aggregates surrounding the negatively charged nucleic acid molecules that can easily merge with the bilipid cell membrane and enter the host cell. Positively charged calcium phosphate molecules form a complex with the negatively charged nucleic acid molecules and generate a precipitate that enters the host cells through endocytosis. The calcium phosphate method of transfection does not require special reagents and is inexpensive. However, this method has limited reproducibility with low transfection efficiency that depends on the cell type. Cationic polymers, such as dendrimers, linear or branched poly (ethylene imine) (PEI), poly (L-lysine) and others是阳离子聚合物的例子。一些聚合物ic nanoparticles, solid lipid nanoparticles (SLNP) and inorganic nanoparticles have been used for chemical transfection.¹²

Viral methods

Viral vectors of transfection offer the highest efficiency and can transfect a large variety of cell types. Virus-mediated biological transfection is termedtransduction. Although the term transfection is sometimes used when nucleic acid delivery into host cells is achieved using viral particles, transduction is the correct term that should be used to refer to this process of viral-mediated delivery. Adenoviruses, adeno-associated viruses and retroviruses have been developed for transduction.¹⁶腺病毒是双链DNA病毒can be used to transduce both dividing and non-dividing cells for a short duration. They can elicit strong host immune responses and the experiments with adenoviruses need to be performed in biosafety level 2 laboratories. Adeno-associated viruses (AAV) are single-stranded DNA viruses with an inability to replicate. They induce a weaker immune response in host cells. Retroviruses are RNA viruses that are characterized by the integration of their RNA into the host genome after reverse transcription. This leads to prolonged expression of the gene of interest. Lentiviruses, gammaretroviruses, spumaviruses and alphateroviruses are examples of retroviruses that have been used for biological transfection.¹²

What’s the difference between stable transfection and transient transfection?

Transfection can be classified as stable or transient (Figure 2) depending on the duration of retention of the genetic material in the host cells.¹⁷If the transfected nucleic acids are incorporated into the host DNA or are retained in the host nucleus as an extrachromosomal element, leading to a permanent change in the expression of the desired gene, the process is termed stable transfection. Stable transfection facilitates constitutive expression of genetic material in cell lines and is useful for the generation of clonal cell lines, large-scale protein production applications and also for stable expression during gene therapy.

Figure 2: Diagram representing the differences between stable and transient transfection.

Transient transfection, on the other hand, does not involve the incorporation of the foreign nucleic acid into the host cell genome, resulting in short-term expression of the target genetic material. The nucleic acids are often removed from the cell as a result of environmental perturbation or cell division. Transient transfection is often used to understand the temporary effect of the change in expression on the desired cellular processes.

Here, we describe a generalized lipofection protocol¹⁸for adherent secondary cell lines and primarycell cultureswith plasmid DNA (Figure 3). The quantities of the plasmid DNA and reagents used are applicable for a single well of a 6-well plate and will have to be scaled depending on the size of the culture dish. Lipofection is a relatively low cost, safe, easy and quick method of transfecting cells. While this protocol can be a good starting point, the parameters will have to be standardized andoptimizedbased on the properties of the DNA/RNA as well the host cell type. All procedures are performed under sterile conditions.

Figure 3: Steps involved in transfection of adherent cells using liposomal transfection reagents.

A)Before transfection:

Plasmid DNA: The quality of plasmid DNA is very important for efficient transfection. The gene of interest is usually cloned into an appropriate plasmid DNA backbone downstream from a suitable promoter. A pure and concentrated plasmid DNA preparation is required for transfection.

Plating of cells: The host cells are trypsinized, counted and plated onto an appropriate culture dish in complete culture media 18–24 h before transfection. The cell numbers need to be adjusted so that they reach a confluency of 50–75% at the time of transfection. Care must be taken to avoid contamination and maintain optimal cell health.

B)Transfection:

1. 质粒DNA是稀释100µL的商业vailable, specialized transfection media or serum-free, antibiotic-free cell culture media. The amount of plasmid DNA used will depend on the size of the culture dish in which the cells have been plated and needs to be optimized for the DNA preparation and cell line. It is common to use 1–8 µg of plasmid DNA per well for a 6-well dish.
   
   
2. The commercially available lipofection reagent of choice (2.5–50 µL) is diluted in 100 µL of transfection media or serum-free, antibiotic-free media.
   
   
3. The plasmid and lipofection reagent dilutions are mixed well and incubated for 30 min at room temperature.
   
   
4. After incubation, 800 µL of transfection or serum-free, antibiotic-free media is added to the mix to make up the transfection volume to 1 mL.
   
   
5. The plate containing the cells is removed from the 5% CO₂incubator, the complete culture media removed, each well rinsed with 500 µL transfection media and the transfection mix prepared in the previous step is added gently to the cells. The cells are incubated with the transfection mix for 6–24 h at 37 ℃ in the 5% CO₂incubator.

C) Post-transfection:

The transfection mix is replaced with 3 mL of complete culture media in each well. The cells are incubated for at least 48 h at 37 ℃ in the 5% CO₂incubator. The health of the cells should be monitored regularly.

We have previously described the biological methods of transfection that employ viruses for the delivery of nucleic acids. The term transduction is often used to describe virus-mediated delivery of nucleic acids into host cells. Bacteriophages were first shown to transduce bacterial cells in 1952.¹⁹Since then, viral vectors have been developed to deliver genetic material into host cells by exploiting the natural propensity of certain viruses to transduce cells. Table 1 summarizes the differences between non-viral transfection and transduction.

Table 1:Comparison of transfection and transduction.

Transfection	Transduction
Delivery of foreign nucleic acids using non-viral methods	Delivery of foreign nucleic acids using viral vectors
Gene-transfer efficiency depends on the type of cells, media conditions etc. and is relatively low	Greater gene transfer efficiency
Serum in the media interferes with cellular uptake of nucleic acids	Transduction can be performed in the presence of serum
These methods are relatively harmless to the lab personnel	Viral contamination needs to be carefully handled. Appropriate biosafety measures should be practiced
Often requires specialized equipment and/or special reagents	Relatively easy to perform
Some methods and reagents can be cytotoxic	Viral infection of cells may induce cytopathic effects, such as insertional mutagenesis and immunogenicity
Physical methods, such as electroporation, gene gun and microinjection, and chemical methods, such as lipofection and calcium phosphate transfection, are examples of transfection	Viral transduction is mediated by DNA viruses, such as adenovirus and adeno-associated virus and RNA viruses, such as lentiviruses

Transfection methods have a wide range of applications. Here, a few of them have been briefly described.

Gene therapy:Gene therapy refers to treating genetic diseases by either silencing a defective gene, replacing a defective gene with the corrected version or amplifying the expression of a gene. Over the years, gene therapy has been used to treat diseases such as sickle cell anemia, beta thalassemia, Duchenne’s muscular dystrophy and hemophilia.²⁰

DNA vaccines:DNA vaccines are vaccines that transfect host cells with engineered DNA plasmids to facilitate the expression of recombinant antigensin vivo.²¹These antigens are recognized by the host’s body and stimulate the generation ofadaptive immunity. The entry of DNA plasmids into host cells is achieved throughin vivoelectroporation.

Gene silencing:Transfection of cells with RNA interference (RNAi) molecules such as small interfering RNA (siRNA), which disintegrate the mRNA, or micro-RNA (miRNA), which suppress the translation of the gene of interest, leads to gene knockdown. Gene silencing can also be achieved using the CRISPR/Cas9 system.

Stable cell line generation:Stable transfection is used to generate stable cell lines that express a recombinant protein constitutively. These stable cell lines are extremely useful for large scale production of recombinant proteins. Stable cell lines that express recombinant proteins or have gene knock in/down are often used to study cellular processes andunderstand the structures of proteins²²in laboratories.

Virus production:Viral vectors for applications such as gene therapy involve the insertion of the desired gene into the viral plasmid backbone. The plasmids encoding the different components of the viral vector are transfected into a secondary cell line for assembly and large-scale production of the viruses. Moreover, viral production is employed for the generation of recombinant viruses such as, the influenza A virus, to study the effects of novel mutations and viral strains on the ability of the virus to infect and the efficiency of vaccines.²³

Large-scale protein production:Recombinant proteins have many applications in therapeutics and several monoclonal antibodies, hormones, enzymes and clotting factors are produced as recombinant proteins on an industrial scale.²⁴Further, the rapidly progressing field ofprecision cellular agriculture, which is a sustainable alternative to traditional agriculture, employs transfection as an important step to enable lab-based production of future foods such as, milk,eggsand plant hemoglobin.²⁵Large-scale production of recombinant proteins has been achieved through transfection of recombinant DNA into mammalian cells, bacteria, yeast, plant and insect cells.

Stem cell reprograming and differentiation:Somatic cells can be reprogramed into induced pluripotent stem cells (iPSCs), which can be differentiated into specific cell types by inducing the expression of certain transcription factors. The development of iPSC technology has been possible thanks to the ability to transfect the genes required for reprograming and differentiation of the stem cells. This technology has led to the development of cellular models of human diseases and has immense therapeutic potential.^26,27

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