Transfection is an important technology in molecular biology and cell biology research. It allows us to introduce exogenous genes into cells to conduct gene function research, gene expression regulation, or gene therapy. Conventional transfection techniques can be divided into two categories: transient transfection and stable transfection (permanent transfection).
Transient Transfection
Transient transfection means that foreign genes are introduced into cells and expressed, but the genes are not integrated into the cell genome and therefore do not replicate as the cells grow. Therefore, transient transfection has a limited time, usually lasting only a few days, until the foreign gene disappears due to various factors during cell growth and division. To determine whether cells are successfully transfected, the constructed plasmid contains a reporter group to indicate the presence of the target gene, which can generally be detected two days after transfection.
Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The optimal time interval depends on the cell type, study objectives and specific expression characteristics of the introduced gene, as well as the time required for the reporter gene to reach a steady state. However, within a few days, most of the foreign DNA is degraded by nucleases or diluted by cell division; after a week, its presence is no longer detectable. Transient transfection is most efficient when supercoiled plasmid DNA is used, probably because cells take it up more efficiently.
Stable Transfection
Stable transfection can be established on the basis of transient transfection, but requires an important sporadic process: in a small number of transfected cells, the foreign gene can be integrated into the cell genome. The foreign gene becomes part of the cell genome and is replicated, which is a sign of stably transfected cells. Therefore, stable transfection requires screening of stable cell lines. In the selected cell lines, the plasmid at this time has been completely integrated into the cell genome, replicated as the cells grow, and stably passed on to future generations.
When establishing stably transfected cell lines, we need to use selective markers to distinguish transient transfections from stable transfections. By co-expressing these selectable markers with genes, we can screen out cells in which foreign genes have been successfully integrated into the genome, while eliminating transiently transfected cells. Co-transfection of foreign genes with antibiotic resistance genes (such as neomycin resistance gene neo) is a common method, and then the transfected cells can be screened with the corresponding antibiotics (such as geneticin or G418). Only stably transfected cells will acquire resistance to antibiotics and survive long-term culture, thereby enabling the selection and expansion of target cells.
Differences in Experimental Operations
The plasmids used for transient transfection and stable transfection are different. The plasmid used for transient transfection does not need to contain resistance, while the plasmid used for stable transfection must contain specific resistance to facilitate subsequent clone selection. In addition, the culture media and experimental reagents used by the two are also different.
The operation of transient transfection is simpler than that of constructing a stable cell line. After constructing the plasmid, the target protein can be obtained through cell recovery, transfection, cell culture, protein purification and other steps. To construct a stable cell line, the constructed plasmid needs to be linearized first, then introduced into cultured mammalian cells, and the fusion of the plasmid and cells can be achieved through a certain transfection method. Then, stable transfected cell lines can be obtained through cell pool screening, monoclonal screening, cell subculture and other steps. Cultivation of stable cell lines can produce target proteins stably over a long period of time.
Characteristics and Applications of Transient Transfection and Stable Transfection
|
Category |
Features |
Scope of application |
|
Transient transfection |
1. Rapidly achieve temporary gene expression |
1. Short-term functional studies and experiments to temporarily express needs |
|
Stable transfection |
1. Long-term stable expression of target genes |
1. Long-term functional studies and experiments for stable expression requirements |