Transfection is a general technique for cell culture, which is frequently used in the research and development of a drug. In in-vitro transfection, cargo molecules, such as the RNA and DNA, are delivered to the cultured cells. In in-vitro transfection, cargo molecules, like plasmid DNA and therapeutic siRNA, are delivered to the target tissue. We can find commercial transfection products for in vivo and in-vitro transfection or utilize the transfection services offered by CROs.
Usually, there are multiple transfection methods carried out through techniques like calcium phosphate exposure, electroporation, and liposome-based transfection. In this article, we have discussed the transfection methods utilized in laboratories.
What is Transfection?
In a more refined definition, we use the transfection process to deliver nucleic acids, such as RNA or DNA to the cells; these are methods other than causing a viral infection. Through this type of foreign nucleic acid introduction with the help of physical, chemical, and biological methods, cell properties can be modified. This allows scientists to study protein expression and the gene function of the cell.
The introduction of nucleic acid in the cell is achieved in two ways: either transiently or stably. In the transient method, the nucleic acid is not replicated and it stays for a limited time. In the stable method, nucleic acid integrates into the genome leading to replication.
Methods Used For Transfection
We currently have various chemical, biological, and physical methods to deliver nucleic acids to the cell cultures. However, not every method is suitable for every experiment or cell culture. In fact, the transfection efficiency, normal physiology effects, cell toxicity, and gene expression widely vary in different methods, which are used for different cell cultures.
It is still possible to broadly define all these transfection types in two categories: stable (nucleic acid is present for long-term, it affects the progeny of the cell culture) and transient (nucleic acid is present for some or define time).
Below we have discussed both types of transfection techniques:
Transient Methods
When transient transfection is carried out, the nucleic acid which is introduced in the cell culture only stays for a regulated time. It is not introduced to the genome of the cell, which means that the transfected genetic modifications are not progressed during cell division to generations. In fact, this transfected material can be diluted at the time of the division of cell or lost because of environmental factors. However, the resultant of high transfected genetic material causes expressed proteins to increase, which stays in the cell.
In general, depending on how transgene is expressed, it can be detected for 1-7 days. However, transfected cells can be harvested for 24-96 hours only.
During the analysis, the gene products require protein or RNA isolation so that enzymatic activity immunoassays can be developed. The optimum time for this is dependent on the goal of the research, cell type, characteristics of a specific expression, and the time needed to achieve a stable state. But, as already discussed, the degradation of foreign DNA happens through cell division or nucleases. In a week, the presence of this nucleic acid introduction may not be seen.
The most efficient type of transient transfection is achieved with supercoiled plasmid DNA because it is more efficiently approved by the cell. But, mRNA, miRNA, siRNA, and proteins can also be utilized for this type of transfection.
Stable Methods
The stable transfection method is a bit different than the transient method. In this process, the foreign DNA or nucleic acid remains in the genome of the cell or it remains as an episomal plasmid. In transient transfection, exogenous DNA stays for a week or a little longer. However, stable transfection is not the same. Here, the exogenous DNA remains in the cell and related progeny for the long-term. This means that stable transfection has the power to introduce nucleic acid or gene in generations.
For this reason, stable transfection is seen as suitable for recombinant protein production. It is also used to understand the long-term impact of the DNA expression or for the downstream analysis.
However, only a few copies of this gene or exogenous DNA is introduced in the cell or genome. This means that stable transfection genes are still much lower in amount than transiently introduced genes. This is possible because transfecting stably into the genome is rare. To successfully achieving this, we need proper DNA delivery along with an effective method to select cells that have introduced the DNA.
One way to achieve this is by introducing a selectable marker that was used for a stable transfection process. This can be utilized for a suitable selection process once the recovery period is over.
Usually, these selectable markers become the genes that have resistance to selected drugs or genes which take place of defective genes in the cell after transfection. In this type of medium, the transient cells or not transfected cells die and the stably transfected cells survive.
Another successful method used for selecting stably transfected genes is to screen morphological or phenotypical changes.
Summary
Transfection is utilized to make recombinant proteins or impede (or enhance) gene expression in the cells which are transfected. This is a powerful and extremely valuable tool that helps us study the regulations and functions of genes and gene products. It additionally allows transgenic organism development and enhances the gene therapy process.