pDsRed-Express2-1載體描述 pDsRed-Express2-1 is a promoterless reporter vector that allows the functional analysis of promoters and promoter/enhancer combinations cloned into its multiple cloning site (MCS). The vector encodes 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. 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 sequence immediately upstream of DsRed-Express2 has been converted to a Kozak consensus sequence (4) to enhance translational efficiency in eukaryotic cells. SV40 polyadenylation signals down-stream 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.
pDsRed-Express2-1 is designed to be used as an in vivo reporter of gene expression. Promoters should be cloned into the pDsRed-Express2-1 MCS, located upstream of the DsRed-Express2 coding sequence. Without the addition of a functional promoter, this vector will not express DsRed-Express2.
The pDsRed-Express2-1 vector can be transfected into mammalian cells using any standard transfection method. Mammalian cells expressing DsRed-Express2 (excitation and emission maxima: 554 and 591 nm, respectively) can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection. If required, stable transfectants can be selected using G418. pDsRed-Express2-1載體含有以下元件： MCS (multiple cloning site): 12–89
DsRed-Express2 (Discosoma sp. red fluorescent protein variant)
Kozak consensus translation initiation site: 90–100
Start codon (ATG): 97–99; Stop codon: 772–774
SV40 early polyA+ signals
Polyadenlyation signals: 926–931 & 955–960; mRNA 3' ends: 964 & 976
f1 origin of replication (for packaging the noncoding strand of DsRed-Express2): 1023–1478
SV40 origin of replication: 1819–1957
Kanr/Neor (kanamycin/neomycin resistance gene)
Neomycin phosphotransferase coding sequences: 2003–2797
pUC origin of replication: 3382–4015 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
