pDsRed-Express-1 is a promoterless mammalian expression vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). It encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed). DsRed-Express contains nine amino acid substitutions which improve the solubility of the protein, reduce the time from transfection to detection of red fluorescence, and decrease the level of residual green emission. When DsRed-Express is expressed in mammalian cell cultures, red-emitting cells can be detected by either fluorescence microscopy or flow cytometry 8-12 hours after transfection. Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate. The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells.
The sequence upstream of the DsRed-Express gene has been converted to a Kozak consensus sequence to enhance translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3′ end of the DsRed-Express mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
Promoters should be cloned into the pDsRed-Express-1 MCS upstream from the DsRed-Express coding sequence. Without addition of a functional promoter, this vector will not express DsRed-Express. The recombinant DsRed-Express vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418.
