Classical transfection techniques were originally developed to introduce plasmid DNA into cells, and plasmid DNA is still the most commonly used vector for transfection. DNA plasmids containing recombinant genes and regulatory elements can be transfected into cells to study gene function and regulation, carry out mutation analysis and biochemical characterization of gene products, study the impact of gene expression on cell health and life cycle, and produce proteins on a large scale for purification and downstream applications.
The topology (linear or supercoiled) and size of the vector construct, the quality of the plasmid DNA, and the choice of promoter are the main factors that influence the efficiency of plasmid DNA transfection.
1. Promoter selection
Promoter selection is very important for efficient expression of transfected genes. Although it has little impact on the transfection process itself, it has a subtle impact on the transfection results. The choice of promoter required to obtain high transfection activity depends on the cell line chosen and the protein to be expressed. The CMV promoter achieves high expression activity in most cell types. In BHK-21, CMV promoter activity is higher than other promoters such as SV40 and RSV. However, the constitutive expression levels of these three viral promoters are low in T cell-derived cell lines, such as Jurkat. The addition of PHA-L and PMA to the culture medium after transfection can activate the CMV promoter in Jurkat cells, and PMA alone is sufficient to activate the CMV promoter in KG1 and K562 (human myeloma leukocytes). Expression from the SV40 promoter is enhanced when containing the large T antigen (present in COS-1 and COS-7), which stimulates extrachromosomal synthesis.
Toxic gene products are also an issue when selecting stably transfected cells. When antibiotic resistance gene expression is detrimental to the health of transfected cells, cells expressing this gene will lose their growth advantage, making it impossible to obtain stable transfected clones with constitutive promoters. In this case, inducible promoters can be used to control the timing of gene expression, allowing selection of stable transfectants. Inducible promoters usually require the presence of an inducible molecule such as a metal ion, metabolite, or hormone to function. However, some inducible promoters work in the opposite way, requiring the absence of a specific molecule to induce gene expression.
2. Plasmid Size and Quality
Whether linearized or supercoiled will affect the transfection results: the transfection efficiency of supercoiled plasmids is much higher than that of linear DNA, especially for transient transfections. Linearized DNA transfection has a high integration rate. Transfection will be more difficult if the plasmid is too large. After all, relatively dense and small foreign bodies are more likely to be endocytosed by cells. If your plasmid happens to be relatively large and you are inexperienced, you may have a higher chance of success by choosing a transfection reagent specifically stated to be able to transfer large plasmids. Some transfection reagents will also provide some components that promote DNA condensation, making the DNA to form a transfection complex denser and easier to transfect. The quality of the purified plasmid will also affect the transfection efficiency. Be sure to choose high-quality plasmids.
3. Concentration and Amount of Plasmid DNA
Since plasmid purification is no longer a problem, beginners usually don't care or are even willing to add more DNA. However, it should be noted that if the amount of DNA is too small, the transfection efficiency will not be high, and too much DNA will also reduce the transfection efficiency. Therefore, the preliminary experiment needs to follow the instructions and mix the appropriate amount of plasmid DNA and transfection reagent in a certain proportion. Some transfection reagents require a larger amount of DNA, while some transfection reagents are more efficient and require less DNA.
4. Set Control
Regardless of the transfection method used, it is important to set up control transfections to check the health of the cells, determine if the reporter assay system is working properly, and determine if there are any insertion-related issues. To check that optimal cell growth conditions are being used, include a negative control (no DNA, no transfection reagent). To determine if the reporter gene detection system is working properly, include a positive control (parallel transfection using a well-established transfection method). To determine if there are insertion-related problems, perform transfection with a plasmid that does not contain the gene of interest.