title: New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.
abstract: Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes.
authors.affiliation: Laboratory of Cell Reproduction, Institute of Microbiology CAS, v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Ivana
authors.initials: I
authors.lastname: Malcova
authors.affiliation: Laboratory of Molecular Microbiology, Institute of Molecular Biology SAS, 845 51 Bratislava, Slovakia.
authors.forename: Marian
authors.initials: M
authors.lastname: Farkasovsky
authors.affiliation: Laboratory of Cell Reproduction, Institute of Microbiology CAS, v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Senohrabkova
authors.affiliation: Laboratory of Cell Reproduction, Institute of Microbiology CAS, v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Pavla
authors.initials: P
authors.lastname: Vasicova
authors.affiliation: Laboratory of Cell Reproduction, Institute of Microbiology CAS, v.v.i., 142 20 Prague 4, Czech Republic hasek@biomed.cas.cz.
authors.forename: Jiri
authors.initials: J
authors.lastname: Hasek
mesh_terms: D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D012641:Selection, Genetic; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins
keywords: Integrative cassettes; dominant selectable markers; episomal tagging; fluorescent proteins; live-cell imaging; yeast