A modified tetracycline regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice.

Summary
A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a transactivator fusion protein (tTA) combining the tetracycline repressor and the transcriptional activation domain of VP16 (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551). This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes RAG-1 and RAG-2, the autoregulatory system yielded both substantially higher levels of V(D)J recombination activity (70 fold on average) and inducible expression in a much larger fraction of transfected NIH3T3 cells (autoregulatory, 90% vs. constitutive, 18%). In addition, this system allowed the creation of inducible transgenic mice in which expression of a luciferase transgene was induced tens to hundreds fold the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. As seen with the unmodified system, leakiness varies between tissues, though it is higher in the thymus with the autoregulatory system. We detected luciferase activity in brain and liver of day 17 fetal transgene positive mice bred in the absence of tetracycline and in thymus, spleen, cerebellum and lung of 3.5 month old mice bred in the absence of tetracycline. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline regulated gene expression both in cultured cells and in transgenic animals.

Citation
Shockett et al., 1995 Natl. Acad. Sci. USA 92, 6522-6526

Background
Systems for inducible mammalian gene expression have typically encountered limitations such as basal leakiness, toxic or nonspecific effects of inducing agents or treatments, limited cell type applicability and low levels of expression (reviewed in 1). Recently, a novel system was described that overcomes many of these difficulties by placing target genes under the control of a regulatory sequence (tetO) from the tetracycline-resistance operon of Tn10. In bacteria, this short sequence is bound tightly by the tetracycline repressor protein (tetR), and binding is blocked by the antibiotic tetracycline. A hybrid fusion protein, the tetracycline transactivator (tTA), combines the tetR DNA binding domain with the transcriptional activation domain of VP-16, such that when tTA binds to a minimal promoter containing tetO sequences, transcription of the target gene is activated. Tetracycline binding to tTA prevents activation presumably by causing a conformational change in the tetR portion of tTA which blocks binding of tTA to tetO; gene activation is achieved by removing tetracycline.

The primary limitation of this system is difficulty in expressing even moderate levels of the tTA protein (undetectable by western blotting and barely detectable by gel electrophoresis mobility shift assay). The authors speculated that this was due to transcriptional "squelching" by the VP16 transactivator domain leading to death of cells expressing even modest levels of the tTA protein. These results combined with the observation of an apparently low level of expression of an inducible luciferase transgene using this system suggest that inefficiencies in tTA expression may contribute to the difficulty.

By placing the tTA gene under the control of a promoter containing tetO, we have created an autoregulatory tTA expression vector that allows higher levels of tTA expression. We demonstrate here that our strategy permits the creation of highly inducible transfected cells with much greater efficiency than the constitutive system, and furthermore, allows the creation of transgenic mice in which expression of a luciferase reporter gene can be controlled by altering the concentration of tetracycline in the drinking water of the animals. The autoregulated expression of transactivator protein should make the tetracycline system applicable to a wider array of problems requiring inducible mammalian gene expression.

Transgenes
Tet-tTA; Tet-Luciferase

Mouse Strain : B6/C3H,
Phenotype: Normal with inducible luciferase activity in a variety of tissues

Gene expression
By comparison to luciferase protein standards, the luciferase activity that we observe in thymus corresponds to an average of approximately 30 molecules of luciferase per cell. However, we do not know what fraction of cells express luciferase activity or how expression levels vary between expressing cells. Since induction of tTA expression in this system depends upon a low level of leakiness of the tTA transgene, we expect that inducibility will vary with the transcriptional profiles of individual cell types and stages of differentiation. Therefore, per cell calculations of luciferase protein may underrepresent the actual levels induced in individual cells.

We are currently examining sections of thymus, brain and spleen from induced and uninduced mice (by in situ hybridization and hopefully cells by FACS) to determine whether tTA and luciferase expression are activated to high levels in a restricted subset of cells or to low levels in many cells.

Inducible activity in spleen, thymus (highest activity, some leakiness), lung, kidney, cerebellum, cerebrum, lymph nodes and to a lesser extent in liver and heart. Although an unpublished result, we have also observed inducible luciferase expression in pancreas.

Keywords
tetracycline responsive system, inducible gene expression, double transgenic mice

Submitted by: Penny Shockett on March 03, 1996

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