pDsRed-Monomer-N1 is a mammalian expression vector that encodes DsRed-Monomer (DsRed.M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1). DsRed-Monomer contains forty-five amino acid substitutions (listed on page 2). When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection (DsRed-Monomer excitation and emission maxima = 557 nm and 592 nm, respectively). The DsRed-Monomer coding sequence is human codon-optimized for high expression in mammalian cells (2).
DsRed-Monomer is well suited for use as a fusion tag. The multiple cloning site (MCS) in pDsRed-Monomer-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Monomer coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Monomer if they are in the same reading frame as DsRed- Monomer and there are no intervening stop codons. A Kozak consensus sequence is located immediately upstream of the DsRed-Monomer gene to enhance translational efficiency in eukaryotic systems (3). SV40 polyadenylation signals downstream of the DsRed-Monomer gene direct proper processing of the 3' end of the DsRed-Monomer 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 in E. coli.
载体应用
pDsRed-Monomer-N1 can be used to construct fusions to the N-terminus of DsRed-Monomer. If a fusion construct retains the fluorescent properties of the native DsRed-Monomer protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene must be cloned into pDsRed-Monomer-N1 so that it is in frame with the DsRed-Monomer coding sequence, with no intervening in-frame stop codons. The inserted gene must include an initiating ATG codon. Recombinant pDsRed-Monomer-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). pDsRed-Monomer-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Monomer.
The DsRed1-N Sequencing Primer (Cat. No. 632387) can be used to sequence genes cloned adjacent to the 5' end of the DsRed-Monomer coding region.
For Western blotting, the Living Colors® DsRed Polyclonal Antibody (Cat. No. 632496) can be used to recognize the DsRed-Monomer protein. However, to generate optimal results it may be necessary to use a higher concentration of antibody than recommended on the DsRed Polyclonal Antibody Certificate of Analysis.