pDsRed-Express-N1 载体

pDsRed-Express-N1 is a mammalian expression vector that encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed; 1). DsRed-Express contains nine amino acid substitutions (listed on page 2), which improve the solubility of the protein and reduce the time from transfection to detection of red fluorescence (2). In addition, these substitutions reduce the level of residual green emission (2). When DsRed-Express is expressed in mammalian cell cultures, redemitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection (DsRed-Express excitation and emission maxima = 557 nm and 579 nm, respectively). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed- Express displays a reduced tendency to aggregate (2). The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells (3).

The multiple cloning site (MCS) in pDsRed-Express-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Express coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. Sequences upstream of DsRed-Express have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). 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 confers kanamycin resistance to E. coli.

载体应用

pDsRed-Express-N1 can be used to construct fusions to the N-terminus of DsRed-Express. If a fusion construct retains the fluorescent properties of the native DsRed-Express protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed-Express-N1 so that it is in frame with the DsRed-Express coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed-Express-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). pDsRed-Express-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express.