In the field of biology science and gene therapy, gene delivery technology is a key means to introduce exogenous genetic material into cells, which directly affects the results of gene function research, disease treatment and biotechnology application. Virus transduction and transfection are two mainstream gene delivery technologies, each of which has its own characteristics and applicable scenarios. Understanding their differences and advantages is very important to promote gene-related research and clinical application.

This paper compares virus transduction and transfection, introduces their principles, virus vector types and transfection methods, analyzes their applicability in different cell types, and emphasizes that technologies should be selected according to needs to achieve the best research results.

Brief Introduction of Transduction and Transfection

Virus transduction refers to the process of introducing foreign genes into target cells by virus vectors. As a natural gene transmitter, virus has been modified to retain the ability to infect cells efficiently, and at the same time remove the pathogenic genes, making it a safe gene delivery tool. Virus vectors bind to specific receptors on the cell surface, enter the cell through endocytosis or membrane fusion, and then integrate the foreign genes into the genome of the host cell or express them stably in the cytoplasm.

Transfection refers to the process of introducing foreign genetic material into cells by non-viral methods, which is mainly based on chemical, physical or biochemical principles. The obvious advantages of this technology are simple operation process, low cost, instantaneous expression of foreign genes and wide application in cell line experiments. However, in the face of primary cells, stem cells and other cells that are difficult to transfect, the transfection efficiency is often difficult to meet expectations, and foreign genes mostly exist in the cells in a free state, which makes it difficult to integrate into the host genome, which makes the gene expression time limited and the expression stability poor.

Different gene delivery protocols that can be divided into viral-based, non-viral based or combination of both (hybrid) (Chong et al., 2021)

Virus Vector Mediated Gene Delivery

Virus transduction technology is an efficient gene delivery method to introduce foreign genes into target cells by virus vectors, which is widely used in gene therapy, drug research and development, basic biological research and other fields.

Different Virus Vector Types

Lentivirus vector: Lentivirus belongs to retrovirus family, and its genome is single-stranded RNA. Lentiviral vectors can stably integrate foreign genes into the genome of host cells, infect splinter cell and non-splinter cell, such as neuron cells and myocardial cells, and can stably express foreign genes for a long time, so they are suitable for long-term gene function research or gene therapy.

AAV vector: AAV is a single-stranded DNA virus with low immunogenicity and strong tissue specificity. There are many serotypes of AAV, and different serotypes have preferences for different tissues and cells. AAV2 has a high infection efficiency for liver cells, and AAV9 can penetrate the blood-brain barrier to infect central nervous system cells, which has a broad application prospect in gene therapy in vivo.

Adenovirus vector: Adenovirus is a double-stranded DNA virus, which has high infection efficiency and can quickly start gene expression in a variety of cells. However, due to the relatively strong immunogenicity of adenovirus vector, foreign genes usually exist in cells in free form, and the expression time is relatively short, which is often used in short-term gene expression research, vaccine development and some gene therapy schemes that need to produce rapid therapeutic effects.

Gene Delivery Process Based on Viral Vetor

Target gene acquisition: First, the target gene fragment should be obtained by gene cloning and other methods. Genomic DNA or RNA can be extracted from biological samples, and the target gene can be amplified by PCR technology, or the target gene with specific sequence can be obtained by artificial synthesis.

Vector selection and transformation: Select the appropriate virus vector according to the experimental purpose and cell type. The vector was modified to remove the pathogenic genes and keep the key cis-acting elements of the virus, so as to ensure that the virus vector can be correctly packaged in packaging cells and effectively express foreign genes in target cells. Then insert the target gene into a specific position of the vector, usually located downstream of the promoter, to ensure that the target gene can be transcribed and expressed under the drive of the promoter.

Packaging of virus vector: The recombinant virus vector is introduced into the packaging cell line. Packaging cell lines can provide various proteins and enzymes needed for virus packaging, and help recombinant virus vectors to assemble complete virus particles.

Virus purification: Separating and purifying virus particles from the culture supernatant or cell lysate of packaged cells, and removing impurities, unpacked carrier DNA, cell fragments, etc. Commonly used purification methods include ultracentrifugation, density gradient centrifugation, column chromatography, filtration and so on.

Virus titer determination: accurately determine the titer of virus vector, that is, the number of virus particles with infectious ability per unit volume. Commonly used methods include terminal dilution, real-time fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and so on.

Cell infection: The purified virus vector is incubated with the target cell, and the virus enters the cell by combining with the specific receptor on the cell surface. Different viral vectors enter cells in different ways, such as adenovirus entering cells through receptor-mediated endocytosis, while lentivirus entering cells through membrane fusion. After entering the cell, the virus vector releases the target gene it carries, and the target gene is expressed in the cell to realize gene delivery.

Schematic diagram of the mechanisms involved in viral vector-mediated gene therapy (Devaney et al., 2011)

Transfection: A Detailed Discussion

Transfection technology, as an important means of gene delivery, is widely used in biology research and gene therapy. According to the classification of technology, transfection technology can be divided into physical and chemical methods.

Chemical Transfection

Liposome transfection: Cationic liposomes are positively charged and can combine with negatively charged nucleic acids by electrostatic interaction to form liposome-nucleic acid complexes. The complex can interact with the negative charge on the cell membrane, enter the cell through endocytosis, and then release nucleic acid in the cell to realize gene transfection. This method is easy to operate and has relatively low cytotoxicity, and is suitable for many cell lines.

Polymer transfection: Cationic polymers (such as polyethyleneimine, etc.) are used to form complexes with nucleic acids. These polymers can protect nucleic acids from nuclease degradation, and promote cell uptake through interaction with cell membrane, thus delivering nucleic acids into cells.

Schematic representation of a variety of liposome drug carriers' composition (Juszkiewicz et al., 2020)

Physical Transfection

Electroporation: Instantaneous holes are formed in the cell membrane by applying short high-voltage electric pulses, so that nucleic acid molecules can directly enter the cell. This method has a wide range of applications, and can achieve high transfection efficiency for many types of cells, including primary cells and stem cells. However, electroporation will cause certain physical damage to cells, and it is necessary to accurately control the parameters of electric pulses.

Microinjection method: Nucleic acid is injected directly into the nucleus or cytoplasm by micromanipulation technology. This method can achieve accurate transfection of a single cell, which is suitable for some situations that require low transfection efficiency but need to operate on specific cells.

Gene gun method: Gold particles or tungsten particles wrapped with nucleic acid are accelerated by high-pressure gas and directly penetrated into cells or tissues. It is mainly used in plant cells and some animal cells that are difficult to transfect, but it may cause great damage to cells and the equipment is expensive.

The principle of electroporation (Du et al., 2018)

Factors Affecting Transfection Efficiency

Cell type: There are differences in cell membrane structure, physiological state and metabolic characteristics of different cell types, which will affect transfection efficiency. Generally speaking, tumor cell lines are easier to transfect than primary cells, and the transfection efficiency of cells with active division is higher than that of cells at rest.

Quality of nucleic acid: the purity, integrity and configuration of nucleic acid will affect the transfection effect. Plasmid DNA with high purity, non-degradation and supercoiled configuration usually has high transfection efficiency.

Experimental conditions: including cell density, medium composition during transfection, serum concentration, temperature, pH value and so on. For example, in the process of transfection, appropriate cell density can usually achieve higher transfection efficiency, and too high or too low cell density may affect the transfection effect.

Transduction vs. Transfection: Comparison of Applicability

In the field of gene delivery, transfection and transduction, as two core technologies, show significant differences in their applicability due to different cell types and experimental environments.

Primary Cell

Transfection: Primary cells usually have poor tolerance to transfection reagents, and transfection efficiency is often low. This is because the cell membrane structure and physiological characteristics of primary cells are complex, and there are great differences among different types of primary cells, which makes it difficult for chemical transfection reagents such as liposomes to effectively mediate foreign genes into cells. At the same time, physical transfection methods, such as electroporation, have certain effects on some primary cells, but they may also lead to the decline of cell viability due to cell damage, which may affect the subsequent experimental results.

Transduction: Virus transduction is more suitable for primary cells. Viral vectors can make use of their natural infection mechanism to specifically recognize and bind to receptors on the surface of primary cells, and efficiently introduce foreign genes into cells through endocytosis or membrane fusion, and can realize the stable integration and long-term expression of foreign genes in primary cells, which is conducive to the study of gene function and gene therapy in primary cells.

Stem Cell

Transfection: Transfection of stem cells also faces challenges. Stem cells have unique cell cycle and self-renewal characteristics, and are sensitive to external stimuli. Conventional transfection methods may interfere with the normal physiological state of stem cells and affect their pluripotency and differentiation potential. In addition, the culture conditions of stem cells are harsh, and the reagents and operations during transfection may have adverse effects on their culture environment, leading to abnormal cell growth and differentiation.

Transduction: Virus transduction technology can introduce foreign genes into stem cells without affecting their characteristics. Lentiviral vectors can stably integrate genes into stem cell genomes, and realize the stable transmission and expression of foreign genes in the process of self-renewal and differentiation of stem cells, which provides a powerful tool for gene editing, directional differentiation and cell therapy research of stem cells.

In Vivo/In Vitro

Transfection: Transfection technology is widely used at the cell level in vitro, for example, it has the advantages of simple operation and relatively low cost in the study of gene function and protein expression analysis of cell lines. By optimizing transfection conditions, foreign genes can be introduced into different types of cells, and the transient effect of gene expression can be quickly observed. However, when used in vivo, transfection technology faces many challenges, such as the stability, targeting and immunogenicity of transfection reagents in vivo, which limits its large-scale application in gene therapy and tissue engineering in vivo.

Transduction: Virus transduction technology has important applications in vivo and in vitro. In vitro, it can be used to establish a cell line that stably expresses foreign genes and provide a stable cell model for basic research and drug development. In vivo, virus transduction has unique advantages, which can deliver foreign genes to specific tissues and organs through intravenous injection and local injection, and realize efficient gene expression.

Transfection and transduction of H9 and H1 hES cells in vitro with each of the transfection protocols (Cao et al., 2010)

How to Choose in Experimental Design

In the experimental design, the choice of virus transduction or transfection technology needs to consider the experimental purpose, cell type, gene expression time and vector capacity.

Experimental Purpose

Short-term gene expression analysis: If researcher want to quickly observe the transient expression of foreign genes in cells to verify the function of genes, carry out short-term protein production or carry out preliminary gene regulation research, transfection technology is more suitable. The reporter gene was introduced into cells by liposome transfection or electroporation, and its expression fluorescence was observed in a short time to quickly judge the transfection efficiency and the primary function of the gene.

Construction of stable cell lines: If the stable and long-term expression of foreign genes in cells is needed to study the effects of genes on the long-term biological behavior of cells, such as cell differentiation and long-term growth regulation of tumor cells, or to build a cell line that stably expresses foreign genes, virus transduction is usually a better choice. For example, lentivirus transduction can integrate genes into the genome of host cells, realize long-term stable expression, and facilitate long-term experimental observation.

Carrier Capacity

Small fragment gene: For a small fragment of foreign gene (e.g. less than 5kb), both transfection and transduction techniques can handle it well. Suitable liposomes or other transfection reagents can be selected for transfection technology, and various virus vectors in transduction technology can also meet the requirements.

Large fragment genes: To deliver large fragment genes (such as more than 5kb), virus transduction systems such as lentivirus vectors usually have larger vector capacity advantages, which can carry and deliver large fragment genes into cells more effectively and ensure the integrity and function of genes.

Cost and Time

Transfection technology is usually low in cost, transfection reagents are relatively cheap, and complicated virus production equipment and processes are not needed. However, virus transduction requires the construction of virus vectors, virus packaging and purification, which involves more reagents, consumables and equipment, and the cost is high.

Transfection experiment is relatively simple, and it takes a short time from preparing reagents to obtaining transfected cells, which can usually be completed within a few days. Virus transduction needs to construct and produce virus vectors first, which usually takes several weeks and the whole experimental period is long.

Selecting the proper technology according to different research goals

Conclusion

To sum up, virus transduction and transfection technologies have their own advantages and disadvantages, and they play an irreplaceable role in the field of gene delivery. Scientific researchers should choose the appropriate gene delivery technology according to the specific experimental needs in order to achieve the best experimental results and research objectives. With the continuous development and innovation of technology, gene delivery technology will show a broader application prospect in the field of life science research and gene therapy in the future.