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Lenti-X lentiviral systems FAQs
The frequently asked questions listed below relate primarily to general use of lentivirus for gene delivery applications with a focus on Takara Bio's fourth generation lentiviral packaging systems.
What is the cloning/packaging capacity of an HIV-1-based lentiviral vector?
Wild-type lentiviruses contain ~9.7 kb of genome including both LTRs. Artificially creating a genome larger than this will result in unstable viral particles and a dramatic drop in viral titer. For recombinant lentiviruses such as those generated using Lenti-X systems, much of the viral genome has been replaced with other useful sequences such as selection markers or fluorescent proteins, but enough space remains for cloning transgenes. Since each pLVX vector contains different useful sequences, the available space for cloning your transgene varies. To determine the maximum recommended transgene size, consult your pLVX vector map and determine the position of the end of the 3' LTR (the end of the 5' LTR is always at position 1), then subtract that from 9.7 kb. For example:
- The 3' LTR of pLVX-Puro ends at 5.4 kb, so ~4.3 kb of space remains for you to clone in your gene
- The 3' LTR of pLVX-IRES-tdTomato ends at 6.3 kb, so ~3.4 kb of space remains for you to clone in your gene
What fraction of the wild-type HIV-1 genome is present in Takara Bio's lentiviral vectors?
Approximately 25%. Our pLVX-Puro vector carries ~2,430 bp that have a continuous alignment with the 9,719-bp wild-type HIV-1 genome.
Do I need to include an internal poly(A) signal in the expression cassette for my gene of interest (GOI)?
No. The GOI or selection marker expression cassette in the lentiviral vector should not include a poly(A) signal or any other transcription termination signal, since it is located in the 3' LTR of the retroviral vector. A transcription termination sequence in the middle of a lentiviral construct can lead to premature cleavage of viral genomic mRNA, resulting in the loss of downstream sequences that are essential for efficient packaging and transduction of the target cells.
What is the cloning/packaging capacity of a lentiviral vector carrying an IRES fluorescent protein expression cassette?
If the IRES vector includes a fluorescent protein, the optimal size of the GOI has been empirically determined to be 0.7–1.2 kb—definitely no larger than 2.5 kb. If the IRES vector includes an antibiotic resistance gene rather than a fluorescent protein gene, the GOI can be larger than 2.5 kb, since even decreased expression of the antibiotic resistance gene will be sufficient to confer drug resistance for the transduced cells.
Tip: Fluorescent protein expression is less efficient from an "empty" IRES-containing vector. If you do not clone a GOI into the IRES vector, we recommend inserting a 0.5- to 1.2-kb stuffer sequence upstream of the IRES.
Can I use a lentiviral vector to perform regular plasmid transfections?
Yes. You can use a lentiviral vector to perform transfections as you would with a regular plasmid. However, this negates one of the advantages of lentiviral transduction: reproducible integration of individual copies of your transgene. During stable integration, plasmid vectors break randomly and integrate into the cellular genome nonspecifically with respect to the original vector backbone. By contrast, proviral DNA from a lentivirus is integrated into the transduced cells' genome precisely via the 5' and 3' LTRs, ensuring the integrity and linearity of the integrated expression construct.
What types of lentiviral vectors are compatible with Takara Bio's Lenti-X packaging systems?
All commonly used HIV-1-based lentiviral vectors are compatible with Lenti-X Packaging Single Shots.
What kinds of 293 cells are compatible with the Lenti-X Packaging Single Shots?
What size are Lenti-X Packaging Single Shots reactions?
A single Lenti-X Packaging Single Shots reaction is performed in a single 10-cm tissue culture plate.
How much packaged lentivirus can I obtain from a single Lenti-X Packaging Single Shots reaction?
A single Lenti-X Packaging Single Shots reaction, performed in one 10-cm tissue culture plate, can generate up to 107–108 infectious viral units per ml (IFU/ml) of unconcentrated lentiviral supernatant. Other systems' packaging reactions produce far less IFU/ml (Figure 2).
What generation are the Lenti-X Packaging Single Shots?
Lenti-X Packaging Single Shots use a fourth-generation lentiviral packaging mix that delivers the gag and pol genes on separate packaging vectors (Figure 3), which precludes the formation of replication-competent lentivirus. Click here to watch a video describing how the components work together to produce high titer lentivirus.
Why do I have to use Tet System Approved FBS with Lenti-X Packaging Single Shots?
Our fourth-generation packaging system uses a transactivation cascade to generate high expression of the essential viral components. The Tet-Off transactivator activates expression of the gag-pro gene, the tat transactivator, and rev—all from TRE promoters. The tat transactivator, in turn, activates expression of the pol protein located on a separate plasmid. The presence of trace amounts of tetracyclines in your serum can result in a conformational change in the Tet-Off protein, which makes it unable to bind the TRE promoter and significantly impacts viral protein expression, leading to a reduction in viral titer. Only Takara Bio performs a functional test on all lots of FBS to ensure optimum induction with all Tet-Off and Tet-On tetracycline-inducible gene expression systems (Figure 4).
Can I use the Lenti-X Concentrator to concentrate a retrovirus?
Yes. You can use the Lenti-X Concentrator to concentrate retroviruses, utilizing the longer incubation times recommended in the Retro-X Concentrator Protocol-At-A-Glance. Click here to watch a video showing how simple it is to concentrate lentivirus using Lenti-X Concentrator.
What are the benefits of using Lenti-X GoStix Plus to measure lentiviral titer?
Lenti‑X GoStix Plus cassettes provide a fast and simple method for obtaining an accurate viral titer. Traditional methods for measuring titer such as qPCR, ELISA, or functional assays can be fairly lengthy and cumbersome, and are therefore not ideal for monitoring your supernatant to identify an optimal harvest time, or for testing multiple samples. Lenti‑X GoStix Plus enable you to obtain a titer in 10 minutes by simply adding 20 µl of supernatant to the GoStix cassette and then scanning the cassette with your smartphone camera after a brief incubation. The app also records the titer so that you can email yourself the result for record keeping. The chart below compares the time required to measure lentivirus titer with Lenti‑X GoStix Plus relative to traditional methods.
What is MOI?
MOI (multiplicity of infection) is the number of infectious viral units per cell at the time of infection.
What is IFU?
IFU (infectious units) indicates the ability of the virus to infect cells and express protein. You can determine the infectious viral titer (IFU/ml) by measuring the expression of a reporter or a specific protein by flow cytometry, immunostaining, etc.
What is CFU?
CFU (colony forming units) measures the virus' ability to transduce cells and express protein (cells harboring the integrated proviral DNA, which carries an antibiotic resistance gene, survive the presence of the drug and form cell colonies). Lentiviral titer in CFU/ml is determined by counting drug-resistant cell colonies.
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