Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.
Cell Calcium
; 59(5): 228-39, 2016 05.
Article
in En
| MEDLINE
| ID: mdl-26992273
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Calcium
/
Lentivirus Infections
/
Aequorin
/
Endoplasmic Reticulum
/
Neurons
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Cell Calcium
Year:
2016
Document type:
Article