title: Rapid development of genetically encoded FRET reporters.
abstract: To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.
authors.forename: Alen
authors.initials: A
authors.lastname: Piljić
authors.forename: Iñaki
authors.initials: I
authors.lastname: de Diego
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D064550:Death-Associated Protein Kinases; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium