Transient transfection and stable transfection, both of which transfect the gene of interest into specific mammalian cells to express the protein of interest. However, the two methods are different in terms of principle and operation process.
1. Experimental principle:
Transient transfection principle: the exogenous gene is not transfected into the chromosome of the cell but exists on the free vector, so that the expression product of the gene can be obtained in a short time, but with the continuous proliferation of the cell, the exogenous gene will eventually be lost, and the production of recombinant protein can not be continued.
Stable transfection principle: Transfection of foreign genes into cell chromosomes, the target gene will not disappear with cell passaging, and the target protein can be produced stably for a long time. Stable transfection (stable cell line construction) enables long-term, stable production of recombinant proteins.
2. Steps.
1. The plasmid used for transient transfection and stable transfection is different, the transient plasmid does not need to be resistant, and the plasmid used for stable transfection must have specific resistance to facilitate subsequent clone screening. In addition, the media and experimental reagents used in the two are also different.
2. The operation of transient transfection is simpler than that of constructing a stable cell line, and the target protein can be obtained after cell recovery, transfection, cell culture, protein purification and other steps after the plasmid is constructedTo construct a stable cell line, it is necessary to linearize the constructed plasmid, and then enter the cultured mammalian cells, realize the fusion of plasmid and cell through a certain transfection method, and then go through cell pool screening, monoclonal screening, cell subculture and other steps to obtain a stably transfected cell line. Culturing stable cell lines provides long-term stable production of the protein of interest.
3. Features: Transient transfection: It can quickly produce trace to medium amounts of recombinant proteinsLow cost of experiments;A host can carry multiple copies, which is highly efficient.
Stable cell line construction: long-term stable production of target protein;The cost of subsequent protein production is reduced after the stable transfection is obtained;It can perform editing operations such as gene insertion and gene knockout.
Biochemical assay kits