Cignal™ Lenti Renilla Control (luc)

Kit Contents:

Component	Specification	Concentration* (total volume)
Renilla luciferase Control Reporter - 1 tube	Ready-to-transduce lentivirus constitutively expressing Renilla luciferase	≥ 0.8 X 107 TU/ml (250 µl)**
Renilla luciferase Control Reporter - 8 tube	Ready-to-transduce lentivirus constitutively expressing Renilla luciferase	≥ 0.8 X 107 TU/ml (2000 µl)***

* exact titer specified on Certificate of Analysis

** Supplied material provides sufficient lentivirus to transduce 2.5X10⁶ cells at a multiplicity of infection (MOI) of 1 TU per cell (refer to Cignal Lenti Reporter User Manual). 

*** Supplied material provides sufficient lentivirus to transduce 2X10⁷ cells at a multiplicity of infection (MOI) of 1 TU per cell (refer to Cignal Lenti Reporter User Manual).

Storage Conditions: The Cignal Lenti Reporters are shipped on dry ice. Store all tubes at -80  ºC.

Advantage of Using Cignal Lenti Renilla Control

To show the importance of Cignal™ Lenti Renilla Control (luc) comparative reporter assays were carried out with or without Cignal™ Lenti Renilla Control (luc). In the experiment, Cignal Lenti HIF-1 reporter (luc) [1.5X10⁵ TU] transduced around 15,000 HepG2 cells (24 hours before transduction 7,500 cells were plated per well of 96-well plate) with and without Cignal Lenti Renilla control (luc) [3X10⁴ TU]. After 42 hours of transduction, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 48 hours of transduction, cells were treated with 300 µM; of CoCl₂ for 18 hours. Luciferase or dual-Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. The utilization of Cignal Lenti Renilla control along with Cignal Lenti reporter (luc) improves the fold induction change in hypoxia signaling activity, as normalization of firefly luciferase activity with renilla luciferase activity corrects for cell death caused by the treatment.

The biosafety features engineered into these vectors include:

• A deletion in the promoter/enhancer region of the U3 portion of 3'LTR ensures self-inactivation of the lentiviral construct after transduction and integration into the genomic DNA of target cells.
  

• The Cignal Lentiviral vector and plasmids expressing packaging proteins contain no significant areas of homology, thereby minimizing any chance for recombination.
  

• None of the HIV-1 genes (gag, pol, rev) will be expressed in transduced cells, as they are expressed from packaging plasmids lacking packaging signal. Therefore, the lentiviral particles that are generated are replication-incompetent
  

• No virulence genes ( vpr, vif, vpu and nef) are present in the Cignal Lentiviral vector.
  
  

 

Feature	Function
RSV-5' LTR; Hybrid Rous sarcoma Virus (RSV) enhancer/promoter-U5 long terminal repeat	Permits viral packaging and reverse transcription of viral mRNA
Psi; Packaging signal	Allow viral packaging
RRE; Rev response element	Involved in packaging of viral transcript
cppt; Central polypurine tract	Involved in nuclear translocation and integration of transduced viral genome
Renilla (Renilla luciferase)	Act as an internal control for normalization
hPGK; human phosphoglycerate kinase eukaryotic promoter	Permits high-level expression of the mammalian selection marker (puromycin)
PuroR; puromycin resistance gene	Can be used for mammalian selection
SIN/3'LTR; 3' self inactivating long terminal repeat	Modified 3'LTR that allows viral packaging but self inactivates the 5'LTR for biosafety purpose. The element also contains a polyadenylation signal for efficient transcription termination
f1 ori; f1 origin of replication	Origin of DNA replication for bacteriophage f1
AmpR; ampicillin resistance gene	Allows selection of the plasmid in E.coli
CMV promoter	Permits high level expression of reporter gene