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Summary
Transgenic mice technology has allowed investigators to target candidate
oncogenes to specific organs by means of a tissue-specific promoter to understand the role
these oncogenes play. However, studies aimed at deciphering the susceptibility of these
organs to tumorigenesis at different development and endocrine states remain poorly
defined. This is because of the constitutive expression nature of the oncogene which does
not permit the determination of the initiation and progression events of tumorigenesis.
Therefore, it is highly imperative that a system be developed where the temporal
expression of a candidate oncogene can be regulated to render these studies possible.
Experimental approach
We have modified the existing bitransgenic mice system developed by Ornitz and
co-workers[1] to achieve the goal of tightly regulating transgene expression. Our
bitransgenic mice system, like that of others[1-3], comprises two lines of mice: the
transactivators and the targets. The transactivator line contains a genetically-modified
transcription factor(regulator) that can be targeted to a particular organ by means of a
tissue-specific promoter. Taking advantage of the modular nature of transcription
factors[4], we created a chimeric regulator that contains a DNA binding domain from the
yeast transcription factor Gal4[5], an activation domain from herpes simplex viral protein
VP16[6] and a carboxyl-terminal mutant of the progesterone receptor ligand binding domain
responsive to anti-progestins (i.e. RU486) but not to progesterone[7, 8].
The target line contains a target gene of interest placed under the control of four high-affinity Gal4 DNA binding sites to which the activated regulator can bind to and induce target gene expression.
Figure 3: Induction process of the target gene in bitransgenic mice. The two lines can then be crossed to generate bitransgenic mice. In the bitransgenic lines generated, though the regulator is constitutively expressed, it is inactive. When RU486 is added, the regulator is activated and the expression of the target gene is then induced.
To test the functionality of our bitransgenic mice system, we targeted the regulator to the liver by means of the transthyretin promoter and used the human growth hormon(hGH) gene as a reporter. We have successfully demonstrated a 200-fold induction of growth hormone expression in response to RU486 administered in the bitransgenic mice generated[unpublished data]. The levels of RU486 used do not affect endogenous progesterone and glucocorticoid receptor activity.
See figure 4 for the induction of hGH expression in bitransgenic mice given RU486.
With the functionality of our bitransgenic mice system verified, we began to generate inducible cancer models for the breast, lung and pancreas. Our regulatable system have important implications in cancer studies. The ability to regulate the activity of the regulator is an added bonus that our bitransgenic model offers. Though the regulator is expressed constitutively, it is inactive in the absence of RU486. This will allow the target gene (i.e. oncogene) to be expressed at any desired time point in development by the simple addition of RU486 to the bitransgenic mice. Thus, studies designed to determine the effect of a target oncogene on a particular organ at different time points in development and endocrine states are finally possible. In addition, the susceptibility of a particular organ to an oncogene can be assessed at the various time points in development. We can further alter the level of target gene expression by modifying the dosage of the added RU486. This will allow us to determine the threshold level of target transgene expression needed to generate a particular phenotype. Finally, it is noteworthy to mention that the ability to regulate the expression of the target oncogene will also allow us to determine the multi-stage initiation and progression events of oncogenesis, which are not possible by the non-regulatable promoter-driven transgenic mice systems presently available. In this regard, our inducible bitransgenic system will be particularly suited for the understanding of the role of temporal expression of an oncogene in mammary gland tumorigenesis.
Conclusions
We have successfully demonstrated a significant induction of hGH gene expression in
bitransgenic mice given RU486. With the functionality of our regulatable system verified,
we have begun to generate inducible cancer models in transgenic mice. The utility of our
system will allow us to regulate the expression of an oncogene which will prove to be
highly useful in studies aimed at understanding the role of the expressed oncogene at
specific developmental and endocrine states. In addition, we can also delineate the
multi-stage initiation and progression events in tumorigenesis in our ultimate goal of
understanding cancer. The elucidation of the complex phenomena of cancer remains a
daunting but important task and the versatility of our inducible system should prove
exceedingly successful in meeting this challenge.
References:
1] Ornitz, D. M., Moreadith, R. W. and Leder, P. (1991). Binary System for the Regulating Transgene Expression in Mice: Targeting int-2 Gene Expression with Yeast GAL4/UAS Control Elements . Proc. Natl. Acad. Sci. 88:698-702.
2] Khillan, J. S., Deen K. C., Yu, S H.., Sweet, R. W., Rosenberg, M., and Westphal., H. (1988). Gene Transactivation by the TAT Gene of Human Immonodeficiency Virus in Transgenic Mice. Nucleic Acids Res. 16:1423-1430.
3] Byrne, G. W., Ruddle F. H. (1989). Multiplex Gene regulation: a Two-tiered Approach to Transgene Regulation in Transgenic Mice. Proc. Natl. Acad. Sci. 86:5473-5477.
4] Green, S., and Chambon P. (1986). A Superfamily of Potentially Oncogenic Hormone Receptors. Nature 324:615-617.
5] Giniger, E., Varnum, S. M., and Ptashne, M. (1985). Specific Binding of Gal4, a Positive Regulatory Protein of Yeast. Cell 40:767-774.
6] Triezenberg, S. J., Kingsbury, R. C., and McKnight S. L. (1988). Functional Dissection of VP-16, the Transactivator of herpes simplex Virus Immediate Early Gene Expression. Genes Dev. 2:718-729.
7] Vegeto, E., Allan, G. F., Schrader, W. T., Tsai M-J., McDonnell, D. P., and O'Malley, B. W. (1992), The Mechanism of RU486 Anatagonism is Dependent on the Conformation of the Carboxy-terminal Tail of the Human Progesterone Receptor. Cell 69:703-713.
8] Wang, Y., O'Malley B. W. Jr., Tsai, S. Y., and O'Malley, B. W. (1994). A Regulatory system for Use in Gene Transfer. Proc. Natl. Acad. Sci. 91(17):8180-8184.
Keywords:
Temporal regulation, inducible, oncogene, bitransgenic model, cancer
submitted: July 29, 1996
for additional information contact 
Steven S. Chua
tel. (713) 798-6252
FAX (713) 790-1275
(e-mail sc690763@mbcr.bcm.tmc.edu)
