pIRES2-DsRed-Express2 is a bicistronic expression vector that allows the simultaneous expression of your protein of interest and DsRed-Express2 from the same mRNA transcript.The vector is designed to allow efficient flow cytometric detection of transiently transfected mammalian cells expressing either DsRed-Express2 and a protein of interest, or DsRedExpress2 alone (at lower signal intensity).
DsRed-Express2 is a variant of the Discosoma sp. red fluorescent protein, DsRed (1).It retains the fast maturation and high photostability characteristic of its predecessor,DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate,resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells.DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry
Bicistronic expression of the protein of interest and DsRed-Express2 is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site 2 (IRES2), located between the multiple cloning site (MCS) and the DsRed-Express2 coding region. This IRES allows cap-independent translation of DsRed-Express2 from an internal start site at the IRES/DsRed-Express2 junction (4, 5). Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS.
SV40 polyadenylation signals downstream of the DsRed-Express2 coding sequence direct proper processing of the 3' ends of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli,and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (6). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of this cassette allows kanamycin resistance in E. coli.
Use
When cloning into the pIRES2 DsRed-Express2 MCS, your gene of interest must contain an initiation codon (ATG) and a stop codon. Cells expressing the gene of interest can be quickly identified by screening for DsRed-Express2 fluorescence.pIRES2 DsRed-Express2 and its derivatives can be introduced into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 and 591,respectively) can be detected by flow cytometry or microscopy 24 hr after transfection. However, in some cases, up to 48 hr may be required for detection. If required, stable transfectants can be selected using G418. Please refer to the Living Colors® User Manual provided with this vector for additional information on detection of DsRed-Express2.
