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The Tet-Off system makes use of the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the herpes simplex virus. [5]
TetR protein family members are mostly transcriptional repressors, meaning that they prevent the expression of certain genes at the DNA level. These proteins can act on genes with various functions including antibiotic resistance, biosynthesis and metabolism, bacterial pathogenesis, and response to cell stress. [citation needed]
The transactivator gene expresses a transcription factor that binds to specific promoter region of DNA. By binding to the promoter region of a gene, the transcription factor causes that gene to be expressed. The expression of one transactivator gene can activate multiple genes, as long as they have the same, specific promoter region attached.
They took embryonic progenitor cells, transduced those cells with oncogenic ras, along with the tetracycline transactivator (tta) protein to control p53 expression using doxycycline, a tetracycline analog and tetracycline responsive short hairpin RNA (shRNA).
Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Furthermore, a limited number of bacteria acquire resistance to tetracyclines by mutations.
Humans and mice both have around 30,000 protein-coding genes. The number of mouse genes without a corresponding human homologue is less than 1%. 90% of the human and mouse genomes are syntenic. 40% of both human and mouse genomes can be aligned at the nucleotide level. Mice have relatively short gestation periods.
Two independently generated transgenic strains were crossed to produce the TetTag strain. In the first transgenic construct, the tetracycline-controlled transactivator (tTA) protein and a two hour half-life Green Fluorescent Protein (shEGFP) are expressed under the direction of the fos minimal promoter.
Tetracycline inhibits protein synthesis by blocking the attachment of charged tRNA at the P site peptide chain. Tetracycline blocks the A-site so that a hydrogen bond is not formed between the amino acids. Tetracycline binds to the 30S and 50S subunit of microbial ribosomes. [3] Thus, it prevents the formation of a peptide chain. [25]