A novel inducible mammalian expression system

Objectives: Craniofacial dysplasia is a cardinal characteristic of many genetic disorders, including abnormal size and shape of the jaws, frontal bossing and midface hypoplasia. Recent advances in molecular biology have allowed researchers in investigating the etiologic factors for many of these disorders, in which distinct genetic mutations cause abnormal bone growth and skeletal development. We hypothesize that genetic factors deleterious to cells involved in bone formation and growth can adversely affect skeletal development. We will address our hypothesis by restoring cellular function during development in mice suffering from craniofacial anomalies secondary to targeted gene deletions. For this purpose, we have developed a temporally regulated, inducible expression system, GLVP/CrePr. Methods: Based on the Cre-loxP bacteriophage system (Lasko et al. 1992; Orban et al. 1992), the Cre DNA recombinase was fused to a mutated progesterone receptor (Pr) domain (Kellendock et al. 1996), conferring sensitivity of Cre activity to mifepristone (RU-486). Moreover, a mifepristone-inducible transactivator was constructed by fusing the HSV-1 derived VP16 domain to GAL4 and Pr sequences (GLVP; Wang et al. 1994). Both of these systems are characterized by significant basal levels of leakage activity. To overcome this deficiency, we have fused CrePr down stream to a minimal (GAL4)₅ promoter that can be induced by the mifepristone activated GLVP transactivator. Results: In our GLVP/CrePr system, one ligand (mifepristone) regulates Cre activity at the transcriptional and post-translational levels. Our data demonstrate that our system is characterized by nominal levels of basal activity, which can readily be induced following mifepristone administration. Conclusion: This GLVP/CrePr system can regulate the conditional knock-in or knock-out expression of genes in vitro as well as in vivo. Therefore, GLVP/CrePr based inducible expression systems can be proven very useful in experiments where temporal regulation of a gene of interest is required.