pLVX-EF1α-IRES-mCherry is an HIV-1-based, lentiviral expression vector designed to simultaneously and constitutively express a protein of interest and the green fluorescent protein mCherry from a bicistronic transcript in mammalian cells. mCherry is a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively.

Simultaneous expression of a protein of interest and mCherry is made possible by the presence of an encephalomyo-carditis virus internal ribosome entry site (IRES; 2) positioned between the multiple cloning site (MCS) and the mCherry gene. The IRES allows a protein of interest and mCherry to be translated from a single bicistronic mRNA. Stable, constitutive expression of the bicistronic transcript is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after vector integration into the host cell genome (3).

pLVX-EF1α-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 (4), 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 (5). Finally, pLVX-EF1α-IRES-mCherry also contains a central polypurine tract/central termination sequence