pmCherry-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively. Expression of fusion proteins that retain the fluorescent properties of the unmodified mCherry protein can be monitored by flow cytometry and their localization in vivo can be determined by fluorescence microscopy.

The multiple cloning site (MCS) in pmCherry-C1 is positioned downstream of the mCherry coding sequence. A Kozak consensus sequence is located immediately upstream of the mCherry gene to enhance translational efficiency in eukaryotic systems (2). SV40 polyadenylation signals downstream of the mCherry gene and the MCS direct proper processing of the 3' end of the mCherry (or fusion gene) mRNA.

The vector backbone 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. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter (PSV40 e), the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter (PKanr) upstream of the cassette confers kanamycin resistance in E.coli.

载体应用

The gene of interest must be cloned into pmCherry-C1 so that it is in-frame with the mCherry coding sequence, with no intervening stop codons.

The pmCherry-C1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (3). pmCherry-C1 can also be used as a cotransfection marker, as the unmodified vector will express mCherry in mammalian cells.

For Western analysis, either the Living Colors® DsRed Polyclonal Antibody (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the mCherry protein.