pLVX-MetLuc载体描述 pLVX-MetLuc is a promoterless, HIV-1-based, lentiviral vector that allows the functional analysis of different promoters and promoter/enhancer combinations inserted into its multiple cloning site (MCS). The vector encodes Metridia luciferase (MetLuc), a secreted reporter protein that can be easily detected in the medium surrounding the cells.
Metridia luciferase is a naturally secreted luciferase from the marine copepod Metridia longa. The human codon-optimized Metridia luciferase gene encodes a 219 amino acid (24 kDa) polypeptide that includes a 17 amino acid N-terminal signal peptide necessary for secretion (1). When a functional promoter is cloned into the MCS, located immediately upstream of the reporter, Metridia luciferase is expressed and secreted into the surrounding medium, where it is easily detected by the addition of a chemiluminescent substrate.
Lentiviral particles derived from pLVX-MetLuc allow you to monitor the activity of your promoter of interest in virtually any cell type, even primary cells. pLVX-MetLuc 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 and transgene RNA (2), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target 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-MetLuc 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).In addition to lentiviral elements, pLVX-MetLuc contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase promoter (PPGK) for selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
The pLVX-MetLuc vector, available as part of the Lenti-X Ready-To-Glow Secreted Luciferase Reporter System (Cat. No. 631746), can be used to monitor promoter activity in live cells as well as in vivo. A promoter must be cloned into the MCS, located upstream of the Metridia luciferase coding sequence. Without the addition of a functional promoter, the vector will not express MetLuc. Lentiviral particles derived from the vector allow you to monitor your promoter of interest in virtually any cell type, even primary cells. If required, stable transfectants can be selected using puromycin.
Propagation in E. coli
Recommended host strains: DH5α and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: pUC References 1. Markova, S.V. et al. (2004) J. Bio. Chem. 279(5):3212-3217.
2. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892.
3. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.
4. Zennou, V. et al. (2000) Cell 101(2):173–185.