Several evidences suggest that prolonged desensitization of adrenergic receptors in hypertrophic hearts may be a primary factor in initiating the pathological changes that lead to overt heart failure. The main objective of this project is to generate a mouse model of gene therapy for heart failure. We are evaluating if chimeric proteins consisting of b2 adrenergic receptors fused to the transducing protein Gas (b2AR-Gas) can reverse the pathological changes of experimental heart failure when conditionally expressed in myocardiocytes. We are carrying out two types of studies: (a) An in-depth investigation on the signalling features of the chimeric receptor in transfected cell lines, and (b) the generation of inducible transgenic mice. (a) The biochemical and pharmacological properties of the chimera were studied by radioligand binding assays using a variety of adrenergic agonists and antagonists. Our data indicate that the binding affinities of adrenergic ligands for chimeric receptors are identical to those measured for the wild-type. However, the sensitivities of the chimeric receptor to agonists, as determined by the enhancement of [35S]GTPgS binding in the presence of GDP, are significantly increased, compared to wild-type receptors. This was confirmed by studying agonist enhancement of cholera toxin catalysed ADP-ribosylation of Gas, which demonstrated that the fused b2AR can activate the tethered alpha subunit. This indicates that the chimeric receptor can be very effective in transducing adrenergic signals, even if it will be expressed at very low levels in the mouse heart. We have also prepared a number of stable cell lines permanently expressing both wild-type and chimeric receptors. Such cells will be used to investigate the desensitization and recycling properties of chimeric b2AR-Gas proteins. (b) We have also made progress in the construction of the transgenic mice. The gene of b2AR-Gas, under the control of the CMV-Tet Responder Element (TRE), was injected in fertilized oocytes to generate transgenic mice. Fiftyfour mice were screened by dot-blot and southern blot analysis, and four transgenic founders were identified. Each founder was cross-bred with wild-type mice to generate four different lineages of transgenic animals, and the resulting heterozigous transgenic mice from each family were cross-bred to obtain a homozigous transgenic strain. We are now planning to cross-breed this inducible transgenic animal with the MHC-tTA transgenic mice. This strain expresses the tetracycline transactivator under the control of the cardiac promoter of the Myosin Heavy Chain (MHC), and will be supplied by Jackson Laboratories. Such final step will generate a binary transgenic mouse in which the gene of the modified b2AR receptor will be turned off /on in the cardiac muscle by simple addition of doxycycline in the drinking water.
Generation of a conditional mouse mutant model of gene therapy for heart failure
Mattei E;Di Certo MG;
2001
Abstract
Several evidences suggest that prolonged desensitization of adrenergic receptors in hypertrophic hearts may be a primary factor in initiating the pathological changes that lead to overt heart failure. The main objective of this project is to generate a mouse model of gene therapy for heart failure. We are evaluating if chimeric proteins consisting of b2 adrenergic receptors fused to the transducing protein Gas (b2AR-Gas) can reverse the pathological changes of experimental heart failure when conditionally expressed in myocardiocytes. We are carrying out two types of studies: (a) An in-depth investigation on the signalling features of the chimeric receptor in transfected cell lines, and (b) the generation of inducible transgenic mice. (a) The biochemical and pharmacological properties of the chimera were studied by radioligand binding assays using a variety of adrenergic agonists and antagonists. Our data indicate that the binding affinities of adrenergic ligands for chimeric receptors are identical to those measured for the wild-type. However, the sensitivities of the chimeric receptor to agonists, as determined by the enhancement of [35S]GTPgS binding in the presence of GDP, are significantly increased, compared to wild-type receptors. This was confirmed by studying agonist enhancement of cholera toxin catalysed ADP-ribosylation of Gas, which demonstrated that the fused b2AR can activate the tethered alpha subunit. This indicates that the chimeric receptor can be very effective in transducing adrenergic signals, even if it will be expressed at very low levels in the mouse heart. We have also prepared a number of stable cell lines permanently expressing both wild-type and chimeric receptors. Such cells will be used to investigate the desensitization and recycling properties of chimeric b2AR-Gas proteins. (b) We have also made progress in the construction of the transgenic mice. The gene of b2AR-Gas, under the control of the CMV-Tet Responder Element (TRE), was injected in fertilized oocytes to generate transgenic mice. Fiftyfour mice were screened by dot-blot and southern blot analysis, and four transgenic founders were identified. Each founder was cross-bred with wild-type mice to generate four different lineages of transgenic animals, and the resulting heterozigous transgenic mice from each family were cross-bred to obtain a homozigous transgenic strain. We are now planning to cross-breed this inducible transgenic animal with the MHC-tTA transgenic mice. This strain expresses the tetracycline transactivator under the control of the cardiac promoter of the Myosin Heavy Chain (MHC), and will be supplied by Jackson Laboratories. Such final step will generate a binary transgenic mouse in which the gene of the modified b2AR receptor will be turned off /on in the cardiac muscle by simple addition of doxycycline in the drinking water.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
