pIRES2-ZsGreen1載體描述 pIRES2-ZsGreen1 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between an MCS and the Zoanthus sp. green fluorescent protein (ZsGreen; 3, 4)coding region. This design permits both the gene of interest (cloned into the MCS) and the ZsGreen1 gene to be translated from a single bicistronic mRNA. pIRES2-ZsGreen1 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing ZsGreen1 and the protein of interest. This vector can be used to obtain stably transfected cell lines without time-consuming drug and clonal selection.
ZsGreen1 is a human codon-optimized (5) ZsGreen variant that encodes the brightest commercially available green fluorescent protein. The MCS in pIRES2-ZsGreen1 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the ZsGreen1 gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E.coli. The pIRES2-ZsGreen1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
pIRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS should include an initiating ATG codon. Selection of ZsGreen1-positive cells is possible 24–36 hours after transfection by flow cytometry or fluorescence microscopy. pIRES2-ZsGreen1 and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).
Selection of Stable Transfectants
Selectable marker: plasmid confers resistance to G418 (500 μg/ml) Propagation in E. coli Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 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
