Description
pLVX-IRES-ZsGreen1 is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and ZsGreen1 in virtually any mammalian cell type, including primary cells. ZsGreen1 is a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen. The vector expresses the two proteins from a bicistronic mRNA transcript, allowing ZsGreen1 to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry.
Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and ZsGreen1, facilitates cap-independent translation of ZsGreen1 from an internal start site at the IRES/ZsGreen1 junction (1).
pLVX-IRES-ZsGreen1 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (2), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-IRES-ZsGreen1 also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
Use
pLVX-IRES-ZsGreen1 is designed to constitutively coexpress your protein of interest and ZsGreen1 from PCMV IE when transduced into mammalian cells. Before it can be transduced into target cells, the vector must be packaged into viral particles in HEK293T cells, using our Lenti-X™ HTX Packaging System (Cat. Nos. 631247 and 631249). This packaging system allows the safe production of high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including nondividing and primary cells (5).
ZsGreen1 is the brightest commercially available green fluorescent protein. The presence of this protein allows transductants to be visualized by fluorescence microscopy and sorted by flow cytometry with standard FITC filter sets (ZsGreen1 has an excitation maximum of 493 nm and an emission maximum of 505 nm).
