Transfection refers to the introduction of nucleic acid straight into eukaryotic cells by implementing non-viral methods.
Essentially, it’s a gene transfer process that makes use of various physical and chemical methods to facilitate the study of protein as well as gene function within a cellular environment.
When looking to rely on laboratory-based tests for getting transfection services, few things play quite a significant role. Among all others, the important ones include Cell Health, DNA quality, confluency, serum, antibiotics, and others.
In this post, we take a closer look at discussing the factors influencing transfection services that contribute towards establishing efficiency. However, it is to be noted that with transfection methods and reagents, a certain degree of cell death is inevitable.
Read on to gain an effective insight into the factors involved.
Generally speaking, there occurs forty to eighty percent confluency in the case of transfecting cells. There are only a few numbers of cells that can factor in the culture towards poor growth. That too, without establishing an ideal cell to cell contact.
A considerable number of cells leading to contact inhibition, which in turn makes them resistant towards the uptake of foreign DNA. Cells that divide actively are more prone to pick up on introduced DNA compared to the quiescent cells.
An appropriate growth rate is what is recommended for cell growth and further supplemented by serum as well as other additional factors.
As such any contaminated cells like the ones intervened by mycoplasma or yeast should be never considered for transfection.
Nevertheless, when cell health has been compromised, one needs to discard them at once and reseed them from stock, free from any contamination.
Remember, in the event of any instability, the medium needs to be checked for its freshness, as the dearth of all necessary inclusions can significantly deter cell growth.
Mediums used for transfection can contain antibiotics. Nevertheless, the use of cationic lipid reagents can make way for the permeability of cells. Also, they might increase the number of antibiotics that are there which further results in cytotoxicity as well as account for reduced efficiency of the transfection process.
Hence, it is advised to do away with including antibiotics in a medium meant for transfection. For all stable transfections, antibiotics like streptomycin and penicillin shouldn’t be used as they are competitive inhibitors for other selective antibiotics.
To extract the right kind of effect out of lab-based transfection, it is required to keep the passage count to below fifty. Additionally, one needs to be consistent across the lab experiments for the number of passages used.
Note that inherent characteristics of cells can undergo change, following which they might show resistance to similar transfection conditions, even after a string of repetitions. This invariably leads to poor expressions for the gene in transfection.
For optimum transfection results, plasmid DNA should always be free from protein, chemical, RNA, as well as microbial contamination. Depending upon the amount of DNA for transfection, the right amount of DNA to be used differs greatly.
Usually, the serum within the culture medium is what functions to enhance the transfection with DNA. Nevertheless, for transfection that’s cationic lipid-mediated, it is imperative to work with DNA lipid complexes when the serum is not present. This is chiefly because of the serum proteins interfering with the formation of the complexes.
During transfection of cells with RNA, it is recommended to act when the serum is not present to avoid contaminating with RNases. As such, the majority of cells are reported to stay healthy for over several hours when the process is performed in a medium that is free of serum.
Thus, the serum quality can significantly affect the growth of cells and the end-result of transfection. Under ideal lab conditions, it becomes necessary to gain control for variability across a wide range of serums to extract the desired results.
Different cell types have different medium requirements and play an important role across all major transfection experiments. The required information for choosing the most appropriate medium for a particular cell type can be derived from the literature published, or from the cell banks. However, when no information is available, one has to derive a suitable medium empirically.
While doing so, it is to be ensured that the medium used is a fresh one, particularly in case of unstable components as any medium missing on the necessary supplements and components might be harmful to cell growth.
If you are looking to create a stable cell line, you must allow anywhere between two to three days after the transfection procedure expresses the resistance gene before adding the selective antibiotic.
In case you are working with a serum-free medium, you must resort to reduced amounts of antibiotics compared to what one would have done in a medium containing serums to maintain the good health of cells.
For a transfection method to be successful there can be multiple factors to consider. Although there are multiple strategies involved, the ideal approach should be based on the cell type and desired results.