How do we get DNA from mammalian cells?
Several methods are available of transferring DNA into cells of higher eukaryotes: transfection with calcium phosphate, fusion of cells with liposomes or bacterial protoplasts, and micro-injection of DNA. Transient and/or permanent expression of foreign genes can be obtained in biochemically transformed cells.
What are two ways to examine gene expression in mammalian cells?
Broadly speaking, gene expression can be measured at the RNA level and, for protein-coding genes, at the protein level. RNA levels can be assessed by RT-qPCR, RT-PCR, RNA-seq, Northern blotting, SAGE, DNA microarrays, tilling arrays and in situ hybridization.
How do plasmids transfect cells?
By performing a process of DNA transfection, a plasmid which contains a gene of interest is efficiently delivered to the cells of interest. Upon delivery to the cells plasmid DNA reaches the nucleus during cell division, the gene of interest is transcribed and its transient expression is achieved.
Why are mammalian cells used in biotechnology?
Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification.
Is electroporation transient transfection?
The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. Furthermore, because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined.
What is stable cell transfection?
Stable transfection refers either to the permanent expression of the gene of interest through the integration of the transfected DNA into the nuclear genome, or the maintenance of a transfected plasmid as an extra chromosomal replicating episome within the cell.
What is a plasmid transfection?
Plasmids are small circular DNA molecules that naturally occur in bacteria, and are actually used by the bacteria to transfer genetic information. The mechanism of adding a DNA plasmid into a mammalian cell is known as plasmid transfection.
What is mammalian cell?
In general, mammalian cells are eukaryotic cells. That means that each cell itself is much more complex than a bacterial cell. Moreover, since mammals consist of billions of individual cells in many organs and tissues, there is a lot of specialization among mammalian cells.