It is important to have an estimation of the concentration of infection-forming units (IFUs) in the supernatant, as this measurement will determine how much supernatant needs to be used to achieve a desired multiplicity of infection (MOI), the number of virions per cell in a transduction.
Non-functional methods include p24 ELISA and qRT-PCR measurement of viral genomic RNA and Northern blotting. ELISA and qRT-PCR methods are quick and easy, and are highly consistent between viral preps. Non-functional methods, however, tend to overestimate infectious titer because of free p24 protein, viral RNA, and immature or empty particles present in all packaging supernatants. Therefore, non-functional titers should be used based on previous knowledge of how p24 or genome measurement relates to infectivity.
Functional titer (infectious titer) only measures infectious mature virus; titer measurement is expressed as infectious units per ml (IFU/ml) or transducing units per ml (TU/ml). Functional methods include assessment of the number of colony forming units following antibiotic selection, or, if the vector contains a fluorescent protein, flow cytometry analysis of transduced cells. If the vector does not express a fluorescent protein, determining the number of integrated proviral DNA copies per cell by qPCR is the fastest, easiest, and the best method for assessing functional titer.
The presence or absence of different vector sequence elements (e.g., LTRs, WPRE, cPPT/CTS, etc.) can affect packaging efficiency and thereby viral titer. Our pLVX vectors are optimized for both titer and expression, and will produce higher titers than other vectors. The Lenti-X Packaging System is likely to produce the highest titer for any HIV-1-based lentiviral vector. When using a vector system other than Takara Bio's, be sure to evaluate whether permission from any third party is required for your intended use.
Due to homologous sequences in the LTRs, all lentiviral vectors have a propensity for recombination in certain strains of E. coli. Use strains with a low recombination frequency (e.g., Stellar Competent Cells). Use diagnostic restriction enzyme digests to ensure vector integrity after propagation in E. coli.
Your gene may affect the titer if it alters the normal growth of 293T cells (e.g., a toxic gene). Also, if the gene is very large, the increased distance between the LTRs may reduce packaging efficiency. Wild-type lentiviruses have a 9.7-kb genome from the start of the 5' LTR to the end of the 3' LTR; creating constructs containing an insert larger than this can result in unstable viral particles and a reduction in viral titer. Also, poly A signals should be avoided in order to eliminate the chance of premature truncation of the viral genomic RNA during packaging.
Collect virus 24–48 hours after the media change that follows packaging transfection. Virus titers will typically be highest 48 hours after the start of transfection. You can use Lenti-X GoStix to determine if the supernatant has sufficient lentivirus, or if you should wait longer before harvesting.
Cultures of healthy cells at an early passage provide the best titers. We recommend the Lenti-X 293T Cell Line.
shRNAs have been reported to reduce titers by as much as 30-fold, due to shRNA-mediated self-cleavage of the RNA genome. However, we have not observed this phenomenon with the Lenti-X System, and we routinely generate titers of 108 IFU/ml with our pLVX-shRNA vectors.
We strongly recommend using NucleoBond Xtra Maxi gravity columns to purify "transfection-grade" transfer vector DNA. Our protocol requires 7 µg of transfer vector DNA applied to the Lenti-X Packaging Single Shots.
The Lenti-X Packaging Single Shots uses Tet-Off transactivation; packaging must be performed under tetracycline-free conditions, otherwise the activity of the Tet-Off transactivator will be reduced, resulting in lower titers.
Xfect Transfection Reagent is recommended for transfection of Lenti-X 293T cells. We routinely achieve >99% transfection efficiency in Lenti-X 293T cells when using our optimized reagents.
The Lenti-X Maxi Purification Kit will remove transduction inhibitors, and result in up to a 10-fold concentration of virus, depending on the starting supernatant volume.
Improving cell-virus contact
Depending on the cell type, consider using RetroNectin reagent. The RetroNectin bound virus (RBV) protocol increases colocalization of viral particles and cells while removing transduction inhibitors from conditioned media. RetroNectin reagent is the transduction enhancer of choice for suspension cells and VLA-4- and VLA-5-expressing cells including T cells, B cells, monocytes, NK cells, eosinophils, bone marrow monocytic cells, lymphoid progenitors, thymocytes, activated T-cells, and mast cells.
Another option, Lenti-X Accelerator can be used to speed up transduction, thereby limiting the exposure of sensitive cells to virus-containing conditioned media and Polybrene. Traditional lentiviral transductions require overnight incubation of cells and virus with Polybrene, a transduction enhancer. But with Lenti-X Accelerator, transduction is complete in only 30 minutes without the use of Polybrene.
Ecotropic Receptor Booster can be used to temporarily elevate the density of the ecotropic receptor protein mCAT-1 on the surface of any target cell that you wish to infect with ecotropic virus. This allows, for example, efficient transduction of human cells with ecotropic retrovirus or lentivirus that could otherwise only infect rodent cells. The technology can be used to increase infection efficiency of any cell type that is resistant to viral infection.
Finally, to maximize cell-virus contact, we recommend a brief centrifugation step during infection (i.e., spinfection). Spinfection can improve tranduction efficiency by 2–10 fold. Refer to the Lenti-X Expression System User Manual for additional details.
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