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The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. [3] The Tet-Off system for controlling expression of genes of interest in mammalian cells was developed by Professors Hermann Bujard [] and Manfred Gossen at the University of Heidelberg and first published in 1992.
Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells with a chemical delivery agent like calcium phosphate (CaPi) or polyethyleneimine (PEI). [1]
Gal4 is a modular protein consisting broadly of a DNA-binding domain and an activation domain. The UAS to which GAL4 binds is CGG-N 11-CCG, where N can be any base. [6] Although GAL4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription activator in a variety of organisms such as Drosophila, [7] and human cells, highlighting that the same ...
Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
Vectors used for E. coli expression can be used in this system although specifically designed vectors for this system are also available. Eukaryotic cell extracts may also be used in other cell-free systems, for example, the wheat germ cell-free expression systems. [41] Mammalian cell-free systems have also been produced. [42]
A version called mPB was created by optimizing codon usage for mammalian (mouse) with a 20x increase in activity, [9] and further mutation screening generated hyPB with 10x the activity of mPB. [10] PiggyBac system have been successfully employed to express large genetic sequences, such as a doxycicline-inducible CRISPR interference system. [11]
Transiently transfected mammalian cells are used in this system to find protein-protein interactions. [27] [28] Using a mammalian cell line to study mammalian protein-protein interactions gives the advantage of working in a more native context. [5] The post-translational modifications, phosphorylation, acylation and glycosylation are similar.
In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo.It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 μ plasmid of baker's yeast ...