Transfection of Cell Lines

Transfection of Cell Lines

Dr. Vijay

PhD, Department of Veterinary Microbiology , Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar

Keywords: Biotechnology, Transfection, Cell Lines, GMO, Transformation

1. Introduction

Transfection is a procedure that introduces foreign or recombinant nucleic acid (RNA or DNA) into eukaryotic cells to produce genetically modified cells. Introduction of such nucleic acid can cause a change in the properties of the cell. The introduced genetic materials exist in the cells either stably or transiently depending on the nature of the genetic materials.

• Stable transfection: The introduced genetic material are integrated into the host genome resulting in a sustained transgene expression (Fig. 1a). This process is required when long-term expression of the sequence is desired.A selection marker is generally used to identify cells that have successfully integrated the sequence of interest.
• Transient transfection: In contrast with stable transfection, transient transfection results in expression for a limited period of time as there is no integration of foreign nucleic acid into the genome (Fig. 1b). The effect on target gene expression is temporary (24-72 hours for RNA probes, 48-96 hours for DNA probes). These are most commonly used to investigate the short-term impact of alterations in gene and protein expression. Transiently transfected genetic materials can be lost by environmental factors and cell division, so the choice of stable or transient transfection depends on the objective of the experiment.

Fig. 1 Schematic diagrams of two different transfections

(a) Stable transfection. (b). Transient transfection

2. Purpose of transfection

The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells.

• Gene expression: Transfection is most commonly used for the expression of a gene of interest in tissue cultures. Expression of the protein in eukaryotic cells allows the recombinant protein to be produced with proper folding and post-translational modifications required for its function. It can be used in various forms of bioproduction depending upon the transfection strategy. For example, delivery of reprogramming transcription factors enables the generation of induced pluripotent stem cell (iPSC). Stable transfection, on the other hand, provides the means for the bioproduction of various therapeutic molecules.
• Gene inhibition: Another frequent use of transfection is in inhibiting the expression of specific proteins through RNA interference (RNAi). In mammalian cell lines RNA interference occurs through the expression of non-coding microRNAs (miRNAs). Vector-based systems express miRNA precursors or short hairpin RNA (shRNA) precursors that are processed by endogenous machinery to produce miRNAs or shRNAs, respectively, which then act to inhibit gene expression.

3. Methods of Transfection

Many methods of transfection have been developed (Table 1). Each method uses different approaches that must be considered depending on cell type and purpose. The ideal method should have high transfection efficiency, low cell toxicity, minimal effects on normal physiology, and be easy to use and reproducible. For this discussion, the methods are broadly classified into biologically, chemically, and physically mediated methods.

3.1. Biological methods

The most commonly used method in clinical research is virus-mediated transfection, also known as transduction. Virus-mediated transfection is highly efficient and it is easy to achieve sustainable transgene expression in vivo owing to the viral nature of integration into the host genome. For example, retrovirus murine leukemia virus (MLV) has been used as a viral vector to establish sustainable transgene expression in humans. MLV integrates its DNA into the host genome and the integrated DNA is expressed in the host. The integrated MLV DNA replicates as the host genome does. Consequently, it segregates into daughter cells, which enables sustainable transgene expression.

The major drawbacks of virus-mediated transfection are immunogenicity and cytotoxicity of viral vectors as they me cause inflammatory reaction and insertional mutation, which may lead to oncogenesis. Another disadvantage of this method is that a virus package has limited carrying capacity.

Table 1: Transfection methods

3.2. Chemical methods

Chemical transfection methods are the most widely used methods in research and were the first to be used to introduce foreign genes into mammalian cells. Chemical methods commonly use cationic compounds which have positive charge and make complexes with negatively charged nucleic acids. The resulting complexes are attracted to the negatively charged cell membrane and pass through the cell membrane.

Fig 2. Diagram representing Cationic compound mediated gene delivery

The transfection efficiency of chemical methods is largely dependent on different factors such as nucleic acid/chemical ratio, solution pH, and cell membrane conditions. Therefore, this process results have low transfection efficiency compared to other methods. However, the merits of relatively low cytotoxicity, no mutagenesis, no extra-carrying DNA, and no size limitation on the packaged nucleic acid makes them more popular.

3.3. Physical methods

The physical transfection methods are the most recent methods and use diverse physical tools to deliver nucleic acids. These methods are as follows:

• Micro injection: This method directly injects nucleic acid into the cytoplasm or nucleus, but it requires skill, may often lead to cell death, and is very labor-intensive.
• Biolistic particle delivery:This method employs the use of gold particles that conjugate with nucleic acids. The nucleic acid/particle conjugates are then shot into recipient cells at a high velocity (“gene gun”). This method is straightforward and reliable but it requires expensive instruments and causes physical damage to samples.
• Electroporation:It is the most widely used physical method. The exact mechanism is unknown but it is believed that a short electrical pulse disturbs cell membranes and makes holes in the membrane through which nucleic acids can pass. Because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined.
• Laser-mediated transfection: This method, also known as optoporation or phototransfection, uses a pulse laser to irradiate a cell membrane to form a transient pore. When the laser induces a pore in the membrane, nucleic acids in the medium are transferred into the cell because of the osmotic difference between the medium and the cytosol.

4. Conclusion and Summary

Transfection methods are evolving rapidly. Even within a class, many new products and technologies are launched each year with improved efficiency and less cytotoxicity. From the virus-mediated method to laser-mediated method, each method has its own advantages and disadvantages so selection of the best method depends upon the experimenter’s experimental objectives.

In summary, transfection methodology has developed rapidly and diversely. Consequently we now have plenty of options to choose from, fitting well into our experimental or clinical needs. However, as cell research progresses, more advanced transfection technologies are still in demand.

Please follow and like us: