The most critical factor for successful lentiviral transduction is viral titer. This page will help you understand:
- How to measure and evaluate viral titer
- Upstream factors that influence titer
- Downstream tools that can help improve transduction with lower titer sample
It is important to have an estimation of the concentration of infection forming units (IFU) 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.
Functional Titer Versus Non-functional Titer
Titration protocols can be roughly divided into functional and non-functional methods.
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 best method for assessing functional titer.
The Lenti-X qRT-PCR Titration Kit evaluates viral RNA genome copies/ml using a rapid 4 hour qRT-PCR assay. The procedure involves isolation of viral RNA, and absolute quantification by real-time PCR. It is important to note that not all genome copies are infectious, and the actual IFU/ml is orders of magnitude lower. The exact correlation will vary for each construct, packaging system, and packaging conditions used. However once this correlation is established, as long as these factors remain identical, IFU/ml can be calculated from genome copies/ml for the same viral construct.
The Lenti-X p24 Rapid Titer Kit is a p24-based ELISA that can be used to determine viral particles/ml in approximately 4 hours. Similar to the qRT-PCR assay, since each viral particle is not necessarily infectious, the actual IFU/ml will be orders of magnitude lower than the viral particle number. Again, the exact correlation will vary for each construct, packaging system, and packaging conditions used. However once this correlation is established, as long as these factors remain identical, IFU/ml can be calculated from genome copies/ml for the same viral construct.
Lenti-X GoStix also measures p24 protein in the packaging cell supernatants. This rapid test (> 10 minutes) can be used to quickly determine if virus production is within a usable range or for selecting the best time to harvest your virus.
For Lenti-X vectors containing a fluorescent marker, cells can be transduced and the number of fluorescent cells can be determined 24–48 hours after transduction using flow cytometry.
For Lenti-X vectors that contain a selectable marker, cells can be infected with serial dilutions of the virus stock, and stable transductants can be selected using the appropriate antibiotic. Titer is calculated from the number of drug-resistant colonies that develop after selection. In general, titers determined using this method are lower than those determined by flow cytometry due to the extra stress placed on cells during drug selection and to loss of transductants during colony expansion.
Titration of proviruses
Measuring provirus, the viral genome after integration into the DNA of target cells, provides the most accurate functional titer. The transduction efficiency of different cell types can vary widely, so measuring virus titer from packaging supernatant often will not provide an accurate estimate of how much virus is needed to transduce one cell type versus another. For example, HT1080 cells can be transduced with lentivirus more easily than Jurkat cells; thus, using the same amount of virus supernatant will result in far fewer integrated proviruses and lower multiplicity of infection (M.O.I.) for Jurkat cells. To measure the number of integrated virus copies we recommend the Lenti-X Provirus Quantitation Kit.
Under proper conditions, a titer of >108 IFU/ml can be achieved for lentiviral constructs. The cPPT/CTS and WPRE sequences and the wild-type HIV-1 LTRs on the pLVX vectors all contribute to higher titers. In 13 independent packaging reactions performed with 10 different fluorescent protein constructs, the average titer obtained for pLVX vectors packaged using the Lenti-X system was 1.85 x 108 IFU/ml, as determined by flow cytometric analysis of transduced HeLa cells.
If the IFU/ml titer is low, Lenti-X Concentrator can be used to concentrate the viral supernatant up to 100-fold. This method is scalable, easier and faster than ultracentrifugation, and requires no specialized equipment.
If the titer is low, additional troubleshooting should be performed to increase titer and increase the likelihood of successful transduction. To start, evaluate upstream factors that may have led to lower packaging efficiency.
Features of the transfer vector
The presence or absence of different vector sequence elements (e.g., LTRs, WPRE, cPPT/CTS, etc.) can affect packaging efficiency and thereby viral titer. Clontech’s pLVX vectors are optimized for both titer and expression, and will produce higher titers than other vectors. The Lenti-X HTX Packaging System is likely to produce the highest titer for any HIV-1-based lentiviral vector. When using a vector system other than Clontech’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.
Quality and amount of plasmid used for transfection
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 and 36 µl of the Lenti-X HTX Packaging Mix.
The Lenti-X HTX Packaging System 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.
When you have high viral titer (>108 IFU/ml), small volumes of viral supernatant can be used, minimizing introduction of transduction inhibitors that may be present in the conditioned media. If low transduction efficiency results despite high titer, one of the following downstream tools can help improve transduction.
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 Boosters 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.