Cell transfection refers to the technology of introducing foreign molecules into eukaryotic cells. With the deepening of research on gene and protein functions, transfection has become one of the most commonly used technical methods in scientific research experiments. For cell transfection experiments, transfection reagents, transfection methods, and cell status will all affect the efficiency of cell transfection. This article mainly introduces several factors that affect cell transfection efficiency.
1. Transfection Reagent
Transfection reagents and cell lines also need to work well together. Using the same reagent, different cell lines usually have different transfection efficiencies. However, the selection of cell lines is usually based on the needs of the experiment. Therefore, before the transfection experiment, a suitable transfection reagent should be selected based on the experimental requirements and cell characteristics. Each transfection reagent will provide a list of cell lines and literature that have been successfully transfected. Through this information, you can choose the transfection reagent that best suits the experimental design. Of course, the most suitable transfection reagent is efficient, low-toxic, convenient, and cheap.
2. Cell State
Generally, low cell passages (<50) ensure that the genotype remains unchanged. The most suitable cells for transfection are cells that have reached the exponential growth phase after several passages. The cells grow vigorously and are easiest to transfect. Cell cultures can evolve through mutations, gross chromosomal rearrangements, or changes in gene regulation after being stored in the laboratory for months and years. This can lead to changes in cell behavior relevant to transfection. That is to say, the biological properties of cell lines of the same strain will change to varying degrees under different culture conditions in various laboratories, resulting in changes in their transfection characteristics. Therefore, if you notice a decrease in transfection efficiency, you can try transfecting freshly cultured cells to restore optimal results.
3. Confluency
As a general guideline, transfect cells at 40-80% confluence. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Overpopulation of cells can lead to contact inhibition, making cells resistant to uptake of foreign DNA. Actively dividing cells take up incoming DNA better than quiescent cells.
4. Transfection Method
Different transfection reagents have different transfection methods, but most of them are similar. When transfecting, the recommended method for the specific transfection reagent should be followed. However, it should also be noted that due to the different properties of cells cultured in different laboratories, differences in plasmid quantification, and differences in operating techniques, the transfection effects may be different. Optimal transfection conditions should be determined based on the specific conditions of the laboratory.
5. Vector Construction
The construction of the transfection vector (viral vector, plasmid DNA, RNA, PCR product, oligonucleotide, etc.) also affects the transfection results. Therefore, it is also important to select a promoter with suitable composition or regulatable strength. The transfection positive control constructed with the same vector of the empty vector and other genes at the same time can eliminate the interference of toxic effects. Therefore, the quantity of plasmid used for transfection must first be ensured, generally more than 2 μg. Plasmids that are not pure enough or contain bacterial LPS or other substances that are toxic to cells will also affect the transfection efficiency. At this time, the plasmid should be purified and concentrated.
6. Cell Culture Medium
Medium is the most essential element for transient transfection. Different cells require different culture media, serum and other additives. Please refer to the instructions for the transfection reagent used. Cultures must be free from bacterial, mycoplasma, and fungal contamination.
7. Serum
For traditional transfection methods, which require transfection in serum-free medium conditions, serum is believed to reduce the efficiency of transient transfection. For the currently commonly used transfection reagents, the presence of serum will not affect the transfection efficiency, and may even help to improve the transfection efficiency. However, when performing cationic liposome-mediated transfection, it is important that DNA-liposome complexes should be formed in the absence of serum, as some serum proteins can interfere with complex formation. For RNA transfection, how to eliminate potential RNase contamination in serum is worthy of attention. Serum is an additive containing an uncertain composition of growth factors and other cofactors, and its effects on the growth of different cells vary greatly. Changes in serum quality directly affect cell growth and transfection efficiency.
8. Antibiotic
Antibiotics are often added during cell culture to prevent contamination, but these additives may cause trouble for transfection. For example, penicillin and streptomycin are medium additives that affect transfection. These antibiotics are generally non-toxic to eukaryotic cells, but some transfection reagents increase cell permeability, allowing the antibiotics to enter the cells. This may indirectly lead to cell death and result in low transfection efficiency. At present, transfection reagents can be operated with complete media containing serum, antibiotics and other additives throughout the entire process, which is very convenient and saves troubles such as contamination.