pDsRed-Monomer-Golgi encodes a fusion protein consisting of DsRed-Monomer, a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1), and a sequence encoding the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase (GT; 2). This region of human beta 1,4-GT contains the membrane-anchoring signal peptide that targets the fusion protein to the trans-medial region of the Golgi apparatus (3–5).
pDsRed-Monomer-Golgi encodes a fusion protein consisting of DsRed-Monomer, a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1), and a sequence encoding the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase (GT; 2). This region of human beta 1,4-GT contains the membrane-anchoring signal peptide that targets the fusion protein to the trans-medial region of the Golgi apparatus (3–5).
SV40 polyadenylation signals downstream of the DsRed-Monomer-Golgi fusion direct proper processing of the 3′ end of the mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor ) consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) gene allow stably transfected eukaryotic cells to be selected using G418 (7). A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pDsRed-Monomer-Golgi backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
The pDsRed-Monomer-Golgi Vector is designed for fluorescent labeling of the trans-medial region of the Golgi apparatus in mammalian cells. Fluorescence can be observed in living cells by microscopy or flow cytometry. If required, stable cell lines can be selected using G418 (7). Filter sets are available for detection of DsRed-Monomer using conventional epifluoresence microscopy (8).