The Thermo Scientific Pierce Red Firefly Lentiluc Packaging Kit enables stable, high-level luciferase expression in transfection-resistant cell lines including primary and stem cells.
The Pierce Red Firefly Lentiluc Packaging Kit contains the vectors and reagents to create custom lentivirus particles to stably express Red Firefly luciferase in difficult-to-transfect cells for various experiments, including reporter gene assays. The kit includes a promoterless Red Firefly luciferase transfer vector into which the promoter of interest is cloned upstream of the luciferase gene. This vector is then co-transfected into HEK293T packaging cells along with the Trans-Lentiviral Packaging Mix, a solution of five plasmids that each encodes separate viral packaging elements. Replication-incompetent lentivirus particles are then collected from the cell culture supernatant and used to transduce stable Red Firefly luciferase expression in target cells.
Pre-packaged CMV- and TK-Red Firefly LentiLuc Control Particles facilitate stable luciferase expression under the control of a strong or weak promoter, respectively, and are used as normalization controls in combination with custom lentivirus particles or to create stable cell lines that express varying levels of Red Firefly luciferase.
Highlights:
- Robust signal – Red Firefly luciferase gene is optimized for high expression in mammalian systems
- Broad tropism – vesicular stomatitis virus envelope protein (VSVg) enables viral transduction of a wide range of dividing and non-dividing cell types
- Stable expression – puromycin-N-acetyl-transferase gene (Pur) enables puromycin resistance to select for stable expression in mammalian cells
- Safest lentivirus production – separate packaging plasmids and conditional promoter elements ensure that virions are replication incompetent
Includes:
Complete kit contains Red Firefly transfer vector, packaging mix, control vector and packaging cell transfection reagents
Requires:
BSL-2 or enhanced BSL-2 containment cell culture facility and government and institution approval for acceptance and use of lentivirus particles
Applications:
- Reporter gene assays in difficult-to-transfect cells
- Generation of stable cell lines for:
- Small-molecule screening
- Whole-animal imaging
Product Details:
 |
Generating luciferase expression-ready lentivirus using the Thermo Scientific Pierce Red Firefly LentiLuc Packaging Kit.
- Clone reporter of choice into pLenti-MCS-Red Firefly Luc Transfer Vector.
- Co-transfect transfer vector and Trans-Lentiviral Packaging Mix into HEK293T packaging cells.
- Change the media on the following day to low-serum media.
- Harvest virus 64 hours post-transfection.
- Concentrate and/or purify virus if desired.
- Determine viral titer if desired.
- Transduce assay cells with lentivirus particles.
- Add puromycin to select for stable expressers.
- Assay for Red Firefly luciferase expression.
|
Viral-based gene delivery systems are ideal for cells that are difficult to transfect, including primary cells and non-dividing cells, because of the inherent abilities of various viruses to deliver gene products. Many viral delivery systems are available, such as adenoviral, retroviral and lentiviral platforms, and each has its advantages and disadvantages. Of these, lentiviral delivery systems are very attractive for gene delivery, because they provide very high transduction efficiency and are effective with cell types that are resistant to the lipid-based delivery. Lentiviral vectors stably integrate transgenes into the active chromatin of both dividing and non-dividing cells, have low toxicity, provide long-term transgene expression and exhibit broad tropism.
The Pierce Red Firefly LentiLuc Packaging Kit includes the pLenti-MCS-Red Firefly Luc transfer vector that contains a modified form of the firefly luciferase gene that has a red-shifted emission spectrum. The transfer vector is a promoterless cloning vector into which a promoter of interest is cloned to control luciferase expression.
 |
| Thermo Scientific Pierce LentiLuc Transfer Vector elements. The promoter of interest is cloned into the multiple cloning site (MCS) via the indicated restriction enzymes. The plasmid elements packaged into the virus are indicated in green. |
The kit also includes the Trans-Lentiviral Packaging Mix comprised of five viral packaging plasmids that express viral proteins at ratios optimal for lentiviral assembly and production. Co-transfection of the transfer vector and packaging mix into the HEK293T packaging cell line results in efficient production of replication-incompetent lentiviral supernatant that is collected for transduction of target cells. The virions are constructed using the vesicular stomatitis virus envelope protein (VSVg) to confer broad cell tropism, allowing end-users to transduce a wide range of both dividing and non-dividing mammalian cell types.
 |
The Thermo Scientific LentiLuc Packaging System has a superior safety profile compared to other commercially available lentiviral vector systems. With all of the viral genes necessary for packaging (Gag, Pol, Pro, Rev, Tat, Env) spread across five plasmids, the possibility of reverting to replication-competent virus is reduced. As an additional safety precaution, Gag-Pro and Tat-Rev expression is under the control of the conditional tetracycline responsive promoter element (TRE), limiting expression of these viral components strictly to cells expressing the tetracycline transactivator (via the pTLA1-TOFF plasmid). |
The transfer vector also encodes for puromycin-N-acetyl-transferase (Pac) driven by the phosphoglycerate kinase (PGK) promoter to enable puromycin resistance for stable clone selection. The included Precision LentiORF RFP Control DNA is a lentiviral transfer vector for constitutive red fluorescent protein (RFP) expression and can be used in parallel with the pLenti-MCS-Red Firefly Luc transfer vector as a positive control for viral construction and transgene delivery.
Thermo Scientific Pierce CMV- and TK-Red Firefly LentiLuc Particles are pre-made virions that deliver stable luciferase expression under the control of the cytomegalovirus (CMV) and herpes simplex virus (HSV) thymidine kinase (TK) promoter, respectively. Because the promoters vary in activity, these ready-to-use viruses enable high (CMV) or low (TK) luciferase expression. These particles can be used in combination with lentivirus particles expressing other luciferases as a positive control for infection and transgene delivery or to normalize experimental conditions. They can also be used independently to generate cell lines that stably express varying levels of Red Firefly luciferase.
 |
Thermo Scientific Pierce CMV-Red Firefly LentiLuc Particles provide brighter bioluminescence than other viral particles. HEK293 cells plated in a 24-well plate were transduced with Pierce CMV-Red Firefly LentiLuc or Qiagen* Cignal* Lenti Red Firefly Control Particles at the same multiplicity of infection (MOI). Luciferase signal was then assayed 72 hrs post-transduction using 20μL cell lysate and Thermo Scientific Pierce Red Firefly Flash Assay Kit or the respective competitor assay kit. The graph shows that Red Firefly luminosity is greater than that of the competitor Red Firefly luciferase. |
The luciferase gene in the transfer vector is subject to a limited use label license.
References:
Wu X. et al. (2000) Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther. 2, 47-55.
Related Resources:
Review of bioluminescence and the use of luciferase reporters in life science research
Luciferase Reporter Selection Guide
Luciferase Detection Instrumentation Guide
Related Products:
Pierce Firefly Luciferase Flash Assay Kit
Red Firefly Luciferase Reporter Assay Vectors
|
