Institut Pasteur

Centre National De La Recherche Scientifique

Centre Nationale De La Recherche Scientifique

Institute Pasteur

Philippe Brulet

Herv&eacute; Le Mouellic

Valérie Baubet

Hervé Le Mouellic

Julien Barbier

Jordi Molgo

Kelly Rogers

Sandrine Picaud

Rafael Vazquez-Martinez

Sylvie Roux

Cécile Saint Cloment

Laurent Coen

Rosario Osta Pinzolas

Herv&eacute; LeMouellic

Valerie Baubet

Cécile Institut Pasteur Saint Cloment

The invention relates to a method for optical detection of the dynamics of Cain a biological system, said method comprising monitoring the photons emitted by a recombinant Ca-sensitive polypeptide, which comprises or consists of a chemiluminescent protein linked to a fluorescent protein, present in said biological system. In a particular embodiment, said recombinant polypeptide comprises or consists of the Aequorin and GFP linked by a linker allowing chemiluminescence resonance energy transfer (CRET), and optionally comprises a peptidic fragment capable of targeting said recombinant polypeptide into a specific cellular domain or compartment. The present invention also concerns a transgenic non-human animal expressing said recombinant polypeptide sensitive to calcium concentration, in conditions enabling the in vivo monitoring of Cadynamics. In a particular embodiment, the expression and/or localization of said recombinant polypeptide is restricted to a specific tissue, a single-cell type and/or in a particular cellular compartment or domain.

Non-invasive real-time in vivo bioluminescence imaging of local Ca&#xc2;2+ dynamics in living organisms

A hybrid protein (GFP-TTC) comprising the non-toxic proteolytic C fragment of tetanus toxin fused to green fluorescent protein was used to analyze the functional synaptic organization of neural networks. When injected intramuscularly in vivo, the GFP-TTC hybrid protein binds to tetanus neurotoxin receptors and clusters very rapidly to the active neuromuscular junction. Membrane traffic by GFP-TTC at the pre-synaptic level of the neuromuscular junction is strongly and rapidly influenced by exogenously co-injecting neurotrophic factors, such as BDNF, NT-4, and GDNF, but not by NGF, NT-3, and CNTF. The membrane traffic, directly detected using GFP-TTC in vivo, permits methods of analyzing synaptic functioning as well as methods of modulating neuronal transport using neurotrophic factors and agonists or antagonists thereof.

In vivo modulation of neuronal transport

A method for visualizing an active synapse wherein said method comprises: (a) exposing cells forming the active synapse to a biomarker comprising at least fragment C of tetanus toxin and a reporter protein; and (b) visualizing the biomarker; wherein the accumulation of the biomarker into dendritic spines of the cells allows visualization of an active synapse. Also, a method for screening molecules capable of modulating synapse activity is provided. A kit useful for the early diagnosis of neurodegenerative disease comprises a biomarker comprising at least fragment C of tetanus toxin and a reporter protein.

Methods for direct visualization of active synapses

A modified bioluminescent system comprising a fluorescent molecule covalently linked with a photoprotein, wherein said link between the two proteins has the function to stabilize the modified bioluminescent system and allowing the transfer of the energy by Chemiluminescence Resonance Energy Transfer(CRET).

Polynucleotides encoding chimeric GFP-aequorin as bioluminescent Ca++ reporters

The non-toxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. A hybrid protein encoded by the IacZ-TTC gene fusion retains the biological functions of both proteins in vivo, i.e. retrograde transynaptic transport of the TTC fragment and &#x3b2;-gal enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy is useful for the delivery of a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein can also be used to map synaptic connections between neural cells.

Hybrid proteins that migrate retrogradely and transynaptically into the CNS

A modified bioluminescent system comprising a fluorescent molecule covalently linked with a photoprotein, wherein said link between the two proteins has the function to stabilize the modified bioluminescent system and allowing the transfer of the energy by Chemiluminescence Resonance Energy Transfer (CRET).

Chimeric GFP-aequorin as bioluminescent Ca++reporters at the single cell level

Chimeric GFP-aequorin as bioluminescent Ca&plus;&plus; reporters at the single cell level

The present invention relates to a process for targeted replacement of at least a part of an endogenous gene by at least a part of a foreign gene or targeted insertion of at least a part of a foreign gene at a targeted site in an endogenous gene in a cell by homologous recombination. This process includes (A) providing a vector which contains (1) at least a part of the foreign gene which is heterologous with respect to the endogenous gene, (2) a first flanking DNA sequence homologous to a first genomic sequence situated on one side of the part of the endogenous gene to be replaced or the targeted site; and (3) a second flanking DNA sequence homologous to a second genomic sequence situated on the other side of the part of the endogenous gene to be replaced or the targeted site, the foreign gene being located between the first and second flanking DNA sequences and is complementary to the part of the endogenous gene to be replaced; (B) transfecting a cell with the vector; (C) and selecting a transfected cell that contains the foreign gene at the targeted site or where the part of the endogenous gene is to be replaced. This invention also pertains to the vector, and the cell transfected by the vector.

Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made

The present invention relates to a process for targeted replacement of at least a part of an endogenous gene by at least a part of a foreign gene or targeted insertion of at least a part of a foreign gene at a targeted site in an endogenous gene in a cell by homologous recombination. This process includes (A) providing a vector which contains (1) at least a part of the foreign gene which is heterologous with respect to the endogenous gene; (2) a first flanking DNA sequence homologous to a first genomic sequence situated on one side of the part of the endogenous gene to be replaced or the targeted site; and (3) a second flanking DNA sequence homologous to a second genomic sequence situated on the other side of the part of the endogenous gene to be replaced or the targeted site, the foreign gene being located between the first and second flanking DNA sequences and is complementary to the part of the endogenous gene to be replaced; (B) transfecting a cell with the vector; (C) and selecting a transfected cell that contains the foreign gene at the targeted site or where the part of the endogenous gene is to be replaced.

A process for providing a recombinant, heterologous gene in the genome of a eukaryotic cell is provided. In particular, heterologous DNA is inserted into a recipient gene of the eukaryotic cell by homologous recombination. Moreover, a transgenic or chimeric animal comprising cells with DNA inserted into its genome by homologous recombination is disclosed. Further, the method of making these transgenic animal is taught.

Growing community of inventors

Paris, France

Philippe Brulet