Common Issues in Cell Transfection

Cell transfection is a technique used to introduce exogenous nucleic acids (such as DNA or RNA) into cells. It is widely applied in gene expression studies, gene editing (knock-in/knock-out), protein function analysis, and cellular function research. In simple terms, transfection allows researchers to deliver new genetic information into cells and observe its effects on cellular behavior.

In this article, we’ll explore the different types of transfection methods and address common issues encountered during transfection experiments, helping you ensure success in your experiments.

Types of Cell Transfection

• By Method

Chemical Transfection

This method uses chemical reagents (e.g., liposomes, cationic polymers) to encapsulate and deliver nucleic acids into cells. It is simple to perform and suitable for a wide variety of cell types.

Physical Transfection

Physical methods (e.g., electroporation, microinjection, gene gun) temporarily open the cell membrane or directly introduce nucleic acids into cells. These methods are ideal for hard-to-transfect cells but require specialized equipment.

Biological Transfection

This method uses viral vectors (e.g., lentivirus, adenovirus) to deliver nucleic acids into cells. Viral transfection is highly efficient but comes with higher costs and certain biosafety risks.

• By Integration of Exogenous Nucleic Acids

Transient Transfection

In transient transfection, exogenous nucleic acids are introduced into cells and expressed temporarily without integrating into the host chromosome. This method is ideal for experiments requiring rapid data acquisition, such as gene function or expression analysis.

Stable Transfection

In stable transfection, exogenous nucleic acids integrate into the host genome, enabling long-term expression. Although establishing a stable cell line requires more time, it provides a reliable system for production and long-term research.

01 How to Choose the Right Transfection Method?

The three main transfection methods (chemical, physical, biological) each have specific requirements and use cases:

• Physical Transfection: Requires specialized instruments and consumables, as well as optimization of transfection parameters, making it less accessible to most laboratories.

• Chemical Transfection: The most widely used method due to its affordability and simplicity. However, certain cell types are resistant to chemical transfection, resulting in low efficiency.

• Biological Transfection: For cells that are less responsive to chemical transfection, viral transfection can be used. However, it’s important to note that even with viral methods, some cell types remain difficult to transfect effectively.

02 Why Is Transfection Efficiency Low?

Several factors can affect transfection efficiency:

• Cell Type and Condition: Transfection reagents vary in effectiveness depending on the cell type. Additionally, the state of the cells before transfection (e.g., health, density) significantly impacts efficiency.

• Reagent Dosage: Using too much or too little transfection reagent can negatively affect efficiency. Follow the manufacturer’s recommendations and optimize dosage for your target cells.

• Nucleic Acid Quality: Poor-quality DNA or RNA (e.g., degraded or contaminated) can reduce transfection efficiency.

• Transfection Procedure: Issues such as overly long incubation times when preparing transfection complexes or insufficient post-transfection incubation times can also lead to reduced efficiency.

03 Should Transfection Complexes Be Prepared in Serum-Free Medium?

In general, it is recommended to use serum-free medium or reagent-specific components when preparing nucleic acid and transfection reagent dilutions. This is because serum contains proteins that may interfere with the adsorption of nucleic acids by cationic liposomes/polymers, reducing transfection efficiency. However, some commercially available transfection reagents are designed to work in serum-containing media. Always refer to the instructions provided with the transfection reagent to determine whether serum-free medium is necessary for preparing transfection complexes.

04 How to Reduce High Cell Mortality After Electroporation?

• Cell Condition: Ensure cells are in good health before electroporation and free from contamination.

• Cell Density: Use an appropriate cell density; both overly high and overly low densities can affect transfection efficiency and cell survival.

• Electroporation Parameters: Optimize the voltage, pulse duration, and number of pulses to determine the best settings for your target cells.

• Electroporation Buffer: Use a suitable electroporation buffer to ensure effective conductivity while minimizing cellular damage.

05 Why Do All Cells Die After Drug Selection for Resistance Genes?

Several factors could lead to this outcome:

• Drug Application Timing: After transfecting plasmids containing resistance genes, cells require 24-48 hours to express the resistance gene. Adding the selection drug too early can kill the cells before they express sufficient resistance. Generally, it’s recommended to add the drug 48 hours post-transfection, but pre-experiment optimization is advised to confirm the timing.

• Drug Concentration: If the drug concentration is set too high, it may exceed the resistance capacity provided by the gene, leading to cell death even in successfully transfected cells.

• Resistance Gene Issues: Incorrect resistance gene selection, mutations, or loss of the gene during transfection can also result in failure of the resistance mechanism.