pCMV-DsRed-Express2载体描述 pCMV DsRed-Express2 is a mammalian expression vector designed to be used for whole cell labeling or as a marker of cotransfection. In mammalian cells, the vector constitutively expresses DsRed-Express2, a variant of the Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Express2 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. In fact, DsRed-Express2 has been shown to be the best red fluorescent protein for whole cell labeling applications (3). 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. The DsRed-Express2 gene is positioned just downstream of the constitutively active human cytomegalovirus immediate early promoter (PCMV IE). As a result, mammalian cells transfected with this vector will constitutively express the red fluorescent protein. A Kozak consensus sequence (4) has been placed immediately upstream of the DsRed-Express2 coding sequence to enhance translational efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express2 gene direct proper processing of the 3' end of the DsRed-Express2 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 (5). 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 the cassette expresses kanamycin resistance in E. coli.
pCMV DsRed-Express2 can be used for whole cell labeling or as a cotransfection marker. The vector can be transfected into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 nm and 591 nm, respectively) can be detected by fluorescence microscopy or flow cytometry 8-12 hours after transfection. If required, stable transfectants can be selected using G418. After cotransfection with pCMV DsRed-Express2 and an expression construct of interest, cells can also be sorted by flow cytometry to enrich for transfected cells.
Propagation in E. coli
Recommended host strain: DH5α, HB101, and other general purpose strains. Single-stranded DNA production
requires a host containing an F plasmid such as JM109 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
Plasmid incompatibility group: pMB1/ColE1
Excitation and emission maxima of DsRed-Express2
Excitation maximum = 554 nm
Emission maximum = 591 nm References 1. Matz, M. V. et al. (1999) Nat. Biotechnol. 17(10):969-973.
2. Bevis, B. J. & Glick, B. S. (2002) Nat. Biotechnol. 20(1):83–87. Erratum in Nat. Biotechnol. (2002) 20(11):1159
3. Strack, R. L. et al. (2008) Nat. Methods 5(11):955–957.
4. Kozak, M. (1987) Nucleic Acids Res. 15(20): 8125-8148
5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
