How to use a "Lentivirus" as a vehicle for reporter gene transfection of cells.
⇒Lentivirus protocol to make your own luciferase cell lines - Fast and Easy.
Why Viral Transduction?
Labeling cells with a genetically encoded reporter gene (firefly luciferase, red fluorescent protein, transferrin, thymidine kinase, etc.) is the number one choice for in vivo molecular imaging. As opposed to synthetic fluorescent dyes, radioisotopes or magnetic beads etc. which dilute with cell mitosis, a constitutively expressed reporter gene is the only longitudinally quantitative option. Various transfection methods to introduce foreign DNA in cells exist. A virus is nature’s own shuttle of genetic material and therefore viral infection is by far the most efficient transduction method even for resilient cell lines (Kim et al, 2004). Multiple viral delivery systems exist (Adenovirus, Adeno-Associated Virus, Retrovirus, and Lentivirus). However, the lentivirus distinguishes itself with long term, sustained expression of transgenes. Lentiviral vectors are able to stably integrate into quiescent, non-dividing as well as dividing cells without silencing, and without producing an immune response (Dissen et al., 2009). Transgene efficiency of close to 100% has been reported (Peng et al., 2008) and lentiviruses infect a broad array of species (Craigo and Montelaro, 2010). These features allow for fast and easy DNA reporter labeling of immortal cell lines as well as primary cell lines.
Lentivirus - Background information.
Lentiviral vectors are based upon HIV-1. This virus excels in host cell attachment, receptor mediated entry, viral mediated reverse transcription and genome integration. Minimal immune or inflammatory responses or toxicity have been observed (Dissen et al., 2009). Through genetic engineering, safe lentiviral vector systems have been developed to generate replication incompetent viral particles.
- Separation of structural and replication genes necessary to produce viral particles onto multiple plasmids; No single plasmid contains all the genes necessary to produce packaged lentivirus.
- Deletion of virulence and accessory genes such as the Tat gene essential for replication.
- Promoter disabling mutations engineered into the U3 region of the 3’ long terminal repeat render the viral particles self inactivating (SIN), providing additional safety as vectors should not be able to generate full-length vector RNA after viral integration.
Both second generation (3-plasmid) and third generation (4-plasmid) systems are available commercially. Third generation plasmids consist of 3 helper plasmids: a packaging construct, a VSV-G construct and a Rev construct, along with a Tat-independent gne transfer vector.
Lentiviral infection - How does it work?
Lentiviral particle / Pseudovirus - Components.
Lentivirus: Facts and Applications.
- Lentivirus can accommodate large inserts (up to 10kb).
- High viral titer concentrations of 10^9 TU/ml.
- Lentivirus can be pseudotyped.
- Lentivirus can be used to generate stable cell lines.
- Lentivirus permit internal promoters into the transfer vector so that the reporter genes can be expressed as inducible systems (Kim et al., 2004).
- Lentivirus can be used for long-term expression of short hairpin RNA (shRNA) and siRNA in vitro and in vivo.
- Lentivirus can be used for in vivo transient transgenic expression for at least 3 months.
- Lentivirus can be used to label and track tumor cells, stem cells, white blood cells in vivo.
- Lentivirus can be used to deliver gene therapy in vivo.
Lentivirus Transduction Protocol.
For a sample protocol see Lenti-Fire™.
Lentivirus Reagents for in vivo imaging.
Lenti-Fire™ lentivirus protocol and manual for additional info.
Lentivirus - in vivo Imaging References.
