pIRES2-DsRed-Express 载体

pIRES2-DsRed-Express contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and a mutant of the Discosoma sp. red fluorescent protein DsRed-Express coding region (3–5). This permits both the gene of interest (cloned into the MCS) and the DsRed-Express gene to be translated from a single bicistronic mRNA. pIRES2-DsRed-Express is designed for the efficient selection by flow cytometry of transiently transfected mammalian cells expressing DsRed-Express and the protein of interest. This vector can also be used to express DsRed-Express alone or to obtain stably transfected cell lines without timeconsuming drug and clonal selection.

DsRed-Express is a human codon-optimized (4) DsRed variant of Discosoma sp. red fluorescent protein (6). Its coding sequence contains nine amino acid substitutions which improve solubility of the protein and reduce the time from transfection to detection of red fluorescence (exitation and emission maxima = 557 nm and 579 nm, respectively). In addition, these substitutions reduce the level of residual green emission (5). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate (3, 5).

The MCS in pIRES2-DsRed-Express is between the immediate early promoter of cytomegalovirus (P CMV IE ) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neo r ), 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pIRES2-DsRed-Express backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.