Description
pLVX-IRES-mCherry is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and mCherry in virtually any mammalian cell type, including primary cells. mCherry is a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The vector expresses the two proteins from a bicistronic mRNA transcript, allowing mCherry 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 mCherry, facilitates cap-independent translation of mCherry from an internal start site at the IRES/mCherry junction (1).
pLVX-IRES-mCherry 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-mCherry 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-mCherry is designed to constitutively coexpress your protein of interest and mCherry 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™ HT Packaging System (Cat. Nos. 632160 and 632161). 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).
The presence of mCherry allows transductants to be visualized by fluorescence microscopy and sorted by flow cytometry with standard FITC filter sets (mCherry has an excitation maximum of 587 nm and an emission maximum of 610 nm).