title: Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.
abstract: Targeted aequorin-based Ca(2+) probes represent an unprecedented tool for the reliable measurement of Ca(2+) concentration and dynamics in different sub-cellular compartments. The main advantages of aequorin are its proteinaceous nature, which allows attachment of a signal peptide for targeting aequorin to virtually any sub-cellular compartment; its low Ca(2+)-binding capacity; the wide range of Ca(2+) concentrations that can be measured, ranging from sub-micromolar to millimolar; its robust performance in aggressive environments, e.g., the strong acidic pH of the lysosomal lumen. Lentiviral vectors represent a popular tool to transduce post-mitotic or hard-to-transfect cells both in vitro and in vivo. Furthermore, it has great potential for gene therapy. Last generation lentiviral vectors represent a perfect compromise for combining large insert size, ease of production and handling, and high degree of biosafety. Here, we describe strategies for cloning aequorin probes - targeted to the cytosol, sub-plasma membrane cytosolic domains, the mitochondrial matrix, and the endoplasmic reticulum lumen - into lentiviral vectors. We describe methods for the production of lentiviral particles, and provide examples of measuring Ca(2+) dynamics by such aequorin-encoding lentiviral vectors in sub-cellular compartments of hard-to-transfect cells, including immortalized striatal neurons, primary cerebellar granule neurons and endothelial progenitor cells, which provide suitable in vitro models for the study of different human diseases.
authors.affiliation: Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy. Electronic address: dmitry.lim@uniupo.it.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Lim
authors.affiliation: Department of Biomedical Science, University of Padova, Via Bassi 58/B, 35131 Padova, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bertoli
authors.affiliation: Department of Biomedical Science, University of Padova, Via Bassi 58/B, 35131 Padova, Italy.
authors.forename: M Catia
authors.initials: MC
authors.lastname: Sorgato
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Forlanini 6, 27100 Pavia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Moccia
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D016180:Lentivirus Infections; D009474:Neurons / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D000331:Aequorin; D002118:Calcium