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Dipartimento di Anatomia, Farmacologia e Medicina Legale Tel +39 011 670 7747 Fax +39 011 6707732 Principal Investigator
Crepaldi / Assistant Professor
Animal models with conditional expression of a transgene of interest can be generated. This permits spatial and temporal control of the transgene expression. A successful strategy is based on the use of chemically induced transcription factors responsible for the regulated expression of a target transgene. One of the most widely used approaches is the tetracycline-dependent regulatory system developed by Gossen and Bujard (1992). In our lab, this approach has been succesfully used to build an animal model of Tpr-Met driven melanomagenesis. Our next goal is to generate transgenic mice with conditional expression of HGF ligand, useful for physiologic as In the tetracycline based transactivation system the “effector”, the tetracycline transactivator (tTA), can be expressed by tissue specific promoters to provide tissue specificity. The tTA specifically binds both tetracycline and the operator sequences (tetO) of the tet operon, opportunely located upstream of the target “responder” transgene. In the original version of this system (“tet off”), the tTA cannot bind DNA in the presence of tetracycline (inducer), whereas in a modified version (“tet on”) the “reverse tTA” (rtTA) binds DNA only in the presence of the inducer. Mice bitransgenic for the tet operon oncogene and rtTA will express the transgene in specific tissues when the drinking water is supplemented with the doxycycline, whereas mice bitransgenic for the tet operon transgene and tTA will constitutively express the transgene in specific tissues unless doxycycline is provided. The major advantage of this approach is the ability to repeatedly turn on and off the expression. This strategy has been instrumental in demonstrating that tumor viability is dependent on continuous oncogene function in vivo also in the case of Tpr-Met driven RESEARCH TOPICS
At present, we are involved in three major projects: 1. Animal Model of inducible tumorigenesis and metastasis; 2. Transgenic mice with inducible targeted expression of HGF in cardiac muscle; 3. Transgenic mice with inducible targeted expression of HGF in skeletal muscle; 1. Animal models of inducible tumorigenesis and metastasis. We have already produced a
“HGF responder” transgenic mouse. The “response” plasmid that we have used is the bidirectional variant proposed by Baron et al. (1995), that has been designed to monitor the expression system through a “reporter” gene (in our case: enhanced green fluorescent protein, EGFP). This will allow us to directly visualize the efficiency of the system in transgenic mice or in cells derived from them. By crossing this mouse with different tissue specific ”effectors” we will induce the expression of HGF in liver, melanocytes and skeletal muscle, to obtain the development of HGF-driven hepatocarcinoma, melanoma and rhabdomyosarcoma, respectively. Considering the multistage nature of tumorigenesis, we will accelerate HGF-driven tumor or metastasis onset by crossing the bitransgenic mice with a third mouse genetically ablated of a tumor oncosuppressor. By turning off the expression of HGF in the metastasis we will evaluate if this causes metastasis regression, thus testing the hypothesis that metastatic cells continuosly require HGF ligand in vivo for their survival. 2. Transgenic mice with inducible targeted expression of HGF in cardiac muscle. HGF is
a cytokine with cardioprotective action in vivo, mainly due to angiogenic and anti-apoptotic mechanisms. We will cross our “HGF responder” mouse with a line carrying the transactivator under control of α-Myosin Heavy Chain (MCHA-tTA) and able to target tTA expression into cardiomyocytes. These transgenic mice will be used to study the role of HGF in cardiac muscle regeneration, in a series of different inducible cardiopathologies. In particular, the bitransgenic mice will be used to induce experimental ischemia through ligation of descending coronary artery and the effect on molecular and cellular mechanisms, myocardial architecture and function will be evaluated at different times. The unique property of HGF to stimulate cellular motility and the expression of its receptor (Met) in different types of stem cel s suggest that HGF is a cytokine able to recruit stem cells into the injured tissue. Our animal model will help in identifying and characterizing the HGF-targeted stem cells responsible for cardiac regeneration. 3. Transgenic mice with inducible targeted expression of HGF in skeletal muscle
HGF is a cytokine which is essential for the migration of myogenic precursors from the somites during development, and is involved in triggering satellite cell activation during muscle regeneration. By crossing our “HGF responder” mouse with a line carrying the transactivator under control of Muscle Creatine Kinase (Mck-tTA) we will target HGF expression into skeletal muscle. These transgenic mice should provide a powerful tool for assessing the effect of an extra-dose of HGF on the ability of satellite cells to regenerate muscle upon induced injury. We will assess whether HGF can also recruit bone marrow stem cells to tissue damage. Selected publications 1994-present:
CREPALDI T., PRAT M., GIORDANO S., MEDICO E., COMOGLIO P.M.:Generation of a truncated hepatocyte growth factor receptor in the endoplasmic reticulum. J. Biol. Chem. 269:1750-1755, 1994 CREPALDI T., POLLACK A.L., PRAT M., ZBOREK A., MOSTOV K., COMOGLIO P.M.:Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells. J. Cell. Biol. 125:313-320, 1994 DI RENZO M.F., OLIVERO M., KATSAROS D., CREPALDI T., GAGLIA P., ZOLA P., SISMONDI P. , COMOGLIO P.M.: Overexpression of the Met/HGF receptor in ovarian cancer. Int. J. Cancer 58:658-662, 1994 GRIGIONI W.F., FIORENTINO M., D’ERRICO A., PONZETTO A., CREPALDI T., PRAT M., COMOGLIO P.M.: Overexpression of c-met protooncogene product and raised Ki67 index in hepatocellular carcinomas with respect to benign liver conditions. Hepatology 21:1543-1546, 1995 CREPALDI T., GAUTREAU A.,COMOGLIO P.M., LOUVARD D., ARPIN M. :Ezrin is an effector of the HGF-mediated migration and morphogenesis in epithelial cells. J. Cell Biol 138:423-434, 1997 PRAT M., CREPALDI T., PENNACCHIETTI S., BUSSOLINO F., COMOGLIO P.M.: Agonistic monoclonal antibodies against the Met receptor dissect the biological responses to HGF. J. Cel Sci. 111:237-247, 1998 GRISENDI S., ARPIN M., CREPALDI T.: Effect of hepatocyte growth factor on assembly of zonula occludens-1 protein at the plasma membrane. J. Cell Physiol.,176:465-471, 1998 GRISENDI S., CHAMBRAUD B., GOUT I., COMOGLIO P.M., CREPALDI T. Release of FAP68, a novel protein involved in the mTOR/rapamycin-sensitive pathway, couples activation of HGF receptor to translational control. J.Biol.Chem 276:46632-46638. 2001 PRUNOTTO C., CREPALDI T., FORNI P., IERACI A., KELLY R.G., TAJBAKHSH S., BUCKINGHAM M., PONZETTO C. Analysis of Mlc-lacZ Met mutants highlights the essential function of Met for migratory precursors of hypaxial muscles and reveals a role for Met in the development of hyoid arch-derived facial muscles. Dev. Dyn. 231: 582-591, 2004 TAULLI C., ACCORNERO P., FOLLENZI A., MANGANO T., MOROTTI T., SCUOPPO C.,. FORNI P.E., CREPALDI T., CHIARLE R., NALDINI L., PONZETTO C. RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met mediated tumorigenesis. Cancer Gene Therapy, 12:456-63, 2005


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