Inducible Transgenic Systems

Widespread alteration in a gene's expression pattern from the beginning of development may disrupt normal growth to a severity that produces unhealthy animals, which could complicate analyses or even render the animals of no use. Also, compensatory mechanisms may occur during development that mask the effects of the transgene. In these cases, control over the time of transgene expression is necessary. Several systems have been developed to meet that need. The Tet system is the most widely used. Two variations of this system have been devised that differ in whether or not the gene is expressed constitutively and when exposed to an exogenous compound. The original strategy is the Tet-off system (Figure 2). In this system, an additional mouse line is produced to express tetracycline-controlled trans-activator (tTA) in the tissue(s) of interest. tTA is a fusion protein containing a tet repressor from E. coli transposon Tn 10 and the activating domain of virion protein 16 of herpes simplex virus (Gossen and Bujard 1992). The transgenic mouse is made by injecting a construct with the gene of interest behind a tet-op promoter. When crossed with the tTA mouse line, tTA is expressed and activates the tet-op promoter, inducing expression of the transgene. When exposed to tetracycline (Tet) or doxycycline (Dox), a tetracycline analog, Tet/Dox binds tTA, preventing binding to the promoter and functionally inhibiting the transcription of the gene of interest (Shockett et al 1995; Furth et al 1994). A successful example of this inducible strategy can be seen in a conditional model of Huntington disease. The tet-responsive promoter was designed to drive the expression of chimeric mouse/human exon 1 with a 94-CAG repeat. Animals expressing the transgene showed progressively severe clasping of limps and tremors before an early death. Interestingly, with exposure to doxycycline treatment, expression of the transgene carrying the repeats was inhibited and the motor abnormalities reversed (Yamamoto et al. 2000).

The major disadvantage of the Tet-off system is that even if investigators do not want the gene expressed until later in development, the animal must be exposed to Tet or Dox from conception. The prolonged application of Tet or Dox could result in side effects. One example of this is a study where investigators demonstrated impaired spatial memory and fear conditioning in animals after long-term Dox exposure (Mayford et al. 1996). In addition, clearance of Tet or Dox in the tissue types being studied must be carefully considered.

The reverse system, TET-on, eliminates these problems. Rather than using an activator, a mutant form of tTA that is a Tet repressor (rtTA) is used. Expression of rtTA inhibits the transcription of the gene of interest, and upon exposure to Tet or Dox, the repression is lifted, allowing for gene transcription (Gossen et al. 1995). Gene expression can be achieved within a few hours, and complete induction after twenty-four hours can often be observed. Other inducible

FIGURE 2 (See color version on DVD) Tet-off inducible system. A mouse carrying a Tet transac-tivator gene (tTA) driven by a selected promoter is crossed with a transgenic mouse with the gene of interest positioned behind the tetOp promoter. The tTA protein is expressed and binds to the tetOp promoter to induce transcription of the gene. When exogenous Tet (or Dox) is introduced into the mouse, Tet binds to tTA, and transcription is blocked.

FIGURE 2 (See color version on DVD) Tet-off inducible system. A mouse carrying a Tet transac-tivator gene (tTA) driven by a selected promoter is crossed with a transgenic mouse with the gene of interest positioned behind the tetOp promoter. The tTA protein is expressed and binds to the tetOp promoter to induce transcription of the gene. When exogenous Tet (or Dox) is introduced into the mouse, Tet binds to tTA, and transcription is blocked.

systems have been developed using either steroids or inter-feron-a as the triggering agents (Kelly et al 1997; Kuhn et al. 1995).

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