Question

Define an inducible transgenic and given an example of how it works.

Answer 1

ans:

Inducible transgenic mouse model:

Inducible transgenic mouse models allow for the activation of genes in specific cells and tissues at specific times. Expression levels are dependent on the dose of the agent administered.

Tetracycline-Inducible Systems:

The tetracycline-dependent regulatory systems (tet systems) developed in the laboratory of Herman Bujard permit stringent control of gene expression in a wide range of cells in culture, as well as in transgenic animals. The tet systems rely on two components, i.e., a tetracycline-controlled transactivator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner. tTA is a fusion protein containing the repressor of the Tn10 tetracycline-resistance operon of Escherichia coli and a carboxyl-terminal portion of protein 16 of herpes simplex virus (VP16). The tTA-dependent promoter consists of a minimal RNA polymerase II promoter fused to tet operator (tetO) sequences (an array of seven cognate operator sequences). This fusion converts the tet repressor into a strong transcriptional activator in eukaryotic cells.

In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to the tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is termed tet-OFF, because tetracycline or doxycycline allows transcriptional down-regulation. Because tetracycline and its derivatives are not usually present in living animals, exogenous administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo. A mutant form of tTA, termed rtTA, has been isolated using random mutagenesis (8,9). In contrast to tTA, rtTA is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation. This tet system is therefore termed tet-ON.

The two systems function as mirror images and are functionally equivalent when transferred into mice. It should be noted that tet-ON requires higher doxycycline concentrations to be active, compared with the concentration tet-OFF requires to be inactive. This may be of importance when the systems are used in vivo, because the doxycycline may differ among tissues. The advantages of tet-ON, compared with tet-OFF, are that the transgene is not expressed until doxycycline is given to the animals and that upregulation in vivo is faster than downregulation. Hallmarks of the tet systems are the tightness of control, the ability to regulate gene activity in a tissue-specific manner, the doxycycline dose-dependent responses, and the ability to return to a control situation by simply discontinuing doxycycline administration. However, the major disadvantage of the tet systems is that control of the expression of the acceptor construct is often leaky, because of strong positional effects on the tetO minimal promoter. This requires the generation of several acceptor mouse strains to identify those that express the transgene not constitutively but in an inducible manner. Several recently published reports described improvements of the tet systems (transactivators with less toxicity or different ligand sensitivities, bidirectional tetO minimal promoters, and reduced leakiness)

The tet systems have been used in vivo for the inducible expression of several transgenes, encoding, for example, reporter genes, oncogenes, or proteins involved in the signaling cascade. The tTA and rtTA systems are both active in the kidney. In whole-kidney extracts, 1,000- to 10,000-fold activation of luciferase activity was observed. In that case, expression of the tTA and rtTA transactivators was under the control of a strong promoter (cytomegalovirus IE). We are currently analyzing, in such mice, the cell-specific expression of the tTA and rtTA transactivators, as well as the inducible expression of a reporter gene along the nephron. This will permit the specific use of these existing transgenic mice for the inducible expression of transgenes of interest in renal pathophysiologic models. To obtain more specific expression of the tet systems in the kidney, we are currently generating transgenic mice in which expression of the rtTA transactivator is restricted to the collecting duct.