Lenti-Bicistronic-Luc Partial Kit
|Description||Simultaneous expression of two or three genes by a single lentiviral vector is often required for a diverse range of applications. Until now, IRES-based elements were used for these particular applications. However, it has been well documented that the gene down-stream of IRES exhibits only low level transgene expression. In addition, the IRES element is relatively large in size (> 800bp) and its use is often limited due to the lentiviral packaging capability (<5.0kb). So development of a promoter system with bi-directional functionality was needed for simultaneous expression of two or three different genes by a single lentiviral vector. Now, ABM has developed such a system, the bicistronic vector, that is superior to IRES-based two gene expression systems because its smaller size (500bp) and stronger expression signal for both genes (Amendola et al., 2005), and with the addition of a T2A peptide to the tricistronic vector, up to 3 different genes can be expressed from the same lentiviral vector (Tang et al., 2009).|
|Note||NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only. * pricing for blank cloning vectors for commercial customers is 1.5x the listed price|
|Caution||Not for diagnostic use.|
|Unit quantity||1 Kit|
|Promoter||PGK, Mini CMV|
- Lentiviral Vector Amplification Protocol
- Selection-Drug Killing Curve
- Lentivector Packaging Protocol
- Lentivirus Infection Protocol
- Enhanced Lentivirus Safety Features: Replication Incompetency
- Suggested MOI for Common Cancer Cell Lines
- Lentiviral System FAQ
|Is the 10 ml of quantity for lentivirus or vector? I'd like to get a lenti vector expresses both GFP and my gene of interest like neuroglobin.I will use the lenti vector with packaging vectors to make virus in 293T cells, and then to transduce my target cells with the virus. Can you make the vector for me?|
The 10ml is ready-to-use lentivirus. For your second question, we offer custom gene cloning at affordable prices. If you have your gene in your plasmid with a compatible site with our vector, the subcloning will be $450 per construct. An extra cost will be charged if we have to find the gene for you.
|What is the EGFP Lentivirus Map (cat#LV006)?|
It is cloned in a Lenti-His vector using EcoRI and XhoI cut sites. There is a stop codon at the end of the the EGFP gene so the tag is not expressed.
|What is the wave length the GFP gene?|
Our GFP has a excitation of 488 nm and emission of 507 nm.
|About the infection protocol, how many infections can I do with the 10ml preparation?|
Based on the instruction coming with the package, 2ml is need for a 6-well, thus the 10ml is only enough for 5 infections. This is likely to be enough for generating a stable cell line.
|If I transfect or infect with an EGFP construct, when will I begin to observe fluorescence?|
Enhanced green fluorescent protein (EGFP), often used to check transfection efficiency or viral titer, can be visualized in packaging cells 24-48 hours after transfection. In target cells, EGFP is usually detected 48-96 hours after infection, depending on the cell type and infection efficiency.
|What is the source for the luciferase gene on the Lenti-UBC-Luc Lentivirus (Cat#LV051)?|
It is from firefly. Luc2 emission spectrum peaks at 600nm in mammalian cells. The substrate is regular D-luciferin.
|How is the IRES-EGFP construct signal compare to the normal GFP control?|
EGFP signal is much weaker due to IRES elements. The levels of EGFP expression is effected by surrounding genetic elements regardless of the high viral particles.
|Can the expression of the lentiviral vector differ among different cellines (for eg,HeLa,MCF 7,293T etc)|
Yes, like transfection, lentiviral vector transduction efficiency is cell line dependent. 293T will have the highest transduction efficiency.
|Are the WPRE and cPPT elements present?|
Both elements are present in all of our pLenti-III vectors. The WPRE is located immediately after the Puromycin marker (between the Puromycin and the 3'LTR). The cPPT is located immediately before the promoter (UbC/PGK/EF1a), between the 5'LTR and the promoter.
|For Cat# LV152, is the 3'LTR region truncated?|
No, this is a 2nd generation construct and the 3'LTR region is not truncated.
|What is the source for the luciferase gene in the Lenti-PGK-Luc Lentivirus (Cat#LV088)?|
It is from firefly (Photinus pyralis)
|Does LV152 contain regular Luc or Luc2?|
LV152 contains Luc2
|What is the difference between Luc2 and regular Luc?|
Luc2 is an engineered firefly luciferase, which is engineered to remove most cryptic transcription factor binding site and improve mammalian expression.
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