Transfection - RNA / BOC Sciences

What is Transfection?

With the continuous development of molecular biology and cell biology research, cell transfection has become a routine tool for studying gene function in cells. Cell transfection technology can be divided into two main categories: transient transfection and stable transfection.

Transient transfection involves exogenous DNA / RNA, which remains episomal in the host cell, allowing for multiple copies and high-level expression. However, this expression is short-lived, lasting only a few days due to degradation or dilution during cell division. Supercoiled plasmid DNA is preferred for transient transfection due to its higher efficiency in cellular uptake.

Stable transfection allows exogenous DNA to integrate into the host genome or remain as episomal plasmids, passed on to progeny cells. However, only a limited number of copies integrate stably, resulting in lower expression levels compared to transient transfection. To facilitate stable integration, selectable markers are included in DNA constructs. Following a brief recovery period, cells are subjected to appropriate selection pressure.

Schematic diagram of the experimental operation of transfection.

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Common Cell Types Used for Transfection

Cell types commonly used for transfection include primary cells, primary cell derivatives, tumor cell lines, Baby Hamster Kidney cells (BHK-21), Human Embryonic Kidney cells (HEK-293), Human Embryonic Lung cells (HEL), Human Lung Cancer cells (A549), Human Breast Cancer cells (MCF-7), Human Cervical Cancer cells (HeLa), Human Umbilical Vein Endothelial Cells (HUVEC), rat liver cells, neuronal cells, and others.

Methods for Transfection

Viral Transfection

Viral transfection, also known as transduction, employs viral vectors to deliver specific nucleic acid sequences into host cells. Retroviruses like lentivirus are often used for stable transfection, while adenovirus, adeno-associated virus (AAV), and herpes simplex virus are alternative vectors that do not ensure stable transfection. Viral transduction is generally regarded as an efficient method for transfecting challenging cell types, such as primary cells. Retroviruses typically transfect dividing cells, while adenovirus, AAV, and herpes simplex virus can target both dividing and non-dividing cells. However, viral transduction carries a higher risk of cytotoxicity and viral infection compared to non-viral methods.

Non-viral Transfection

Physical/Mechanical Transfection

Non-viral transfection methods based on physical/mechanical approaches can be further categorized into physical/mechanical methods and chemical methods. Common physical/mechanical transfection methods include electroporation, sonoporation, magnetofection, gene microinjection, and laser irradiation.

Chemical Transfection

Chemical transfection can be divided into lipid-based transfection and non-lipid-based transfection. Lipid-based transfection reagents are chemicals that can form positively charged lipid aggregates, which can smoothly fuse with the host cell's lipid bilayer, allowing foreign genetic material to enter with minimal resistance. On the other hand, non-lipid-based transfection reagents can be further categorized into several types, including calcium phosphate, dendrimers, polymers, nanoparticles, and non-liposomal lipids.

Hybrid Methods

Combining viral and non-viral transfection methods is referred to as hybrid methods. Using this combination method generates higher transfection efficiency compared to other transfection methods (e.g., using complexes alone).

What are Applications of Transfection?

Gene Function Research: Introducing exogenous genes into cells through transfection technology, researchers explore the function, expression regulation, and subcellular localization of these genes.

Gene Knockout and Silencing: By transfecting cells with plasmids containing specific gene knockout or silencing sequences, the impact of gene knockout or silencing on cell growth, differentiation, and apoptosis is studied.

Virus Infection Studies: Introducing viral genes into cell lines, researchers investigate the mechanisms of viral infection in cells and the mechanism of action of antiviral drugs.

Cell Differentiation and Fate Determination: Using transfection technology to introduce genes or regulatory elements associated with cell differentiation fate, the processes of cell differentiation, apoptosis, and autophagy are studied.

Vaccine Production: Introducing virus antigen genes into appropriate cell lines, cell lines capable of expressing virus antigens are obtained through transfection technology for vaccine production. For example, introducing virus antigen genes into insect cells can produce efficient and safe viral vaccines.

Development of Gene Therapy: Introducing therapeutic genes (such as therapeutic siRNA, plasmid DNA, etc.) into target cells, gene therapy is achieved through transfection technology. For example, introducing specific genes into myocardial cells can treat diseases such as genetic heart disease or myocardial infarction.

What is the Process of Transfection?

Common Transfection Steps

(1) Cell Preparation: Select appropriate types of cells for transfection and culture them appropriately before transfection to ensure that the cells are in the most favorable state for transfection.

(2) Gene Preparation: Cut and label the target gene in an appropriate manner for subsequent detection and screening.

(3) Preparation of Transfection Vectors: Choose suitable transfection vectors such as liposomes, viruses, etc., and package the target gene into the transfection vector (this step involves vector construction, where the target gene is inserted into the transfection vector to form a recombinant target gene fragment, which is then transfected into target cells using this recombinant fragment to introduce the target gene into the cells).

(4) Transfection: Follow the standard operating procedure provided by the manufacturer to introduce the transfection vector into the recipient cells.

(5) Confirmation of Transfection Efficiency: Confirm whether the target gene has been successfully introduced into the recipient cells through specific detection methods such as fluorescence staining, immunocytochemistry, Western blot, etc.

(6) Screening and Cloning: For cells that need further cloning and amplification, specific screening methods such as antibiotic screening, fluorescence screening, etc., can be used for screening and cloning.

(7) Expansion Cultivation: Expand the screened cells in culture to obtain more cells for subsequent experiments or production.

(8) Functional Verification: After gene transfection, specific functional verification methods such as ELISA, cell viability assays, Western blot, etc., are required to confirm whether the target gene has been successfully expressed and functions.

siRNA Transfection Steps

(1) Design siRNA: Firstly, design siRNA based on the target gene sequence. Online tools or commercial laboratories can be used for design. It is generally recommended to design and validate 2-3 siRNAs to ensure specificity and effectiveness.

(2) Synthesize siRNA: Synthesize the designed siRNA using chemical synthesis methods and purify and concentrate it. Usually, ready-made siRNAs are purchased, but self-synthesized siRNAs can also be used.

(3) Cell Culture: Culture the required recipient cells to an appropriate number and density. Before transfection, ensure that the cells are in the rapid growth phase and in good condition.

(4) Transfect siRNA: According to the instructions of the transfection reagent or laboratory standard operating procedure, transfect siRNA into the recipient cells.

(5) Identification: After transfection, the cells need to be cultured for recovery, and the next step of operation can usually be carried out 24-48 hours later.

miRNA Transfection

miRNA is a non-coding RNA, consisting of about 20-25 nucleotides, which is widely found in eukaryotes and mainly regulates intracellular gene expression. Upon binding to target mRNAs, miRNAs trigger biological processes, such as inhibiting mRNA translation or promoting mRNA degradation. After transfection into recipient cells, miRNAs regulate specific gene expression and affect post-transcriptional gene expression efficiency by binding to target mRNAs. Specifically, miRNAs promote target mRNA degradation and reduce the expression level of specific genes, while inhibiting target mRNA translation and reducing the production of specific gene proteins. These mechanisms enable miRNAs to effectively regulate intracellular gene expression.

mRNA Transfection

mRNA in the cell is responsible for transcribing the genetic information from DNA into readable RNA sequences and delivering them to the ribosomes for protein synthesis. mRNA transfection is the process of introducing externally synthesized mRNA into the cell and translating it into the target proteins using its translation system. The technology is widely used in biomedical fields, such as the development of mRNA vaccines and the production of mRNA drugs. During transfection, mRNA needs to be processed and modified to ensure its stability, protein expression efficiency and correct subcellular localization. These processing steps include 5' cap modification and formation of 3' polyadenylated tails.