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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.
To conduct an inducible lineage-tracing experiment two components must be engineered into the mouse genome: a switch and a reporter. The switch is commonly a drug-regulated form of the bacterial enzyme Cre-recombinase. This enzyme recognizes specific sequences, called LoxP sites. [63]
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]
The GAL4/UAS system is an example of both an inducible and repressible system. Gal4 binds an upstream activation sequence (UAS) to activate the transcription of the GAL1/GAL7/GAL10 cassette. On the other hand, a MIG1 response to the presence of glucose can inhibit GAL4 and therefore stop the expression of the GAL1/GAL7/GAL10 cassette. [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]
[1] [3] This whole system is inducible so a chemical can be added to knock genes out at a specific time. Two of the most commonly used chemicals are tetracycline, which activates transcription of the Cre recombinase gene and tamoxifen, which activates transport of the Cre recombinase protein to the nucleus. [ 4 ]
An expression system is a system specifically designed for the production of a gene product of choice. This is normally a protein although may also be RNA, such as tRNA or a ribozyme . An expression system consists of a gene, normally encoded by DNA , and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA ...
This system has allowed researchers to manipulate a variety of genetically modified organisms to control gene expression, delete undesired DNA sequences and modify chromosome architecture. The Cre protein is a site-specific DNA recombinase that can catalyse the recombination of DNA between specific sites in a DNA molecule.