Flash Therapeutics has developed a proprietary purification platform for lentiviral vectors production. Purification levels are tailored to the target cell type. Flash Therapeutics generates high titer and highly pure lentiviral particles that do not affect cell viability and proliferation.
Lentiviral vectors titer can be measured in a number of ways; some vendors measure the total number of particles in prep, resulting in a titer that includes both functional and non-functional virus particles. The titer information provided by Flash Therapeutics is a functional titer determined by qPCR measurement of only the viral particles that are able to transduce cells (TU; transducing units). Specifically, TU corresponds to the number of integrated genomes generated by transducing 1x105 HCT116 cells with several serial dilutions of viral supernatant.
The lentiviral vectors titer varies for each lot. Please refer to the certificate of Analysis (CoA) for the titer of your particular lot.
Because the titer of each lot of lentiviral particles varies, the volume required for transduction will also vary. The titer can be found on the Certificate of Analysis (CoA) provided with each lentiviral vector batch. Use the formula below to calculate the volume of lentiviral vector for transduction. Please refer to the product user manual for additional information.
Viral vectors volume required (µl) = Number of cells seeded / Viral vectors Titer (TU/ml) x M.O.I x 1000
These products are designated as level 2 biological agents. Follow the biosafety procedures for use of retroviral-derived vectors in accordance with all applicable regulations. All Flash Therapeutics manufactured lentiviral vectors are self-inactivating (SIN). As a result, while the lentiviral vectors maintain the ability to infect a wide range of cell types, they are not capable of producing replicative particles from transduced cells as assessed by RCL/RCR testing. Please refer to the MSDS documents for more information.
Upon receipt, lentiviral vectors must be stored at -80°C.
Just before use, remove the lentiviral vectors from the -80°C freezer and thaw the particles according to the volume of your vial as described below. Once thawed, the vectors should be used for transduction as soon as possible to avoid degradation. Five minutes before transduction, the vial should be removed from ice and allowed to reach room temperature. To avoid any heat shock, allow culture medium to fully equilibrate to room temperature before diluting vectors and cells.
- For a volume of viral vectors higher than 1ml: just before cells transduction, remove the tubes of viral vectors from the -80°C freezer and thaw them using a water-bath at 37°C. Before disappearance of the last ice-cube in the vial, place the viral vectors at room temperature to complete thawing and start the transduction experiment.
- For a volume of viral vectors between 1ml and 100µl: just before cells transduction, remove viral vectors from the -80°C freezer and thaw them at room temperature.
- For a volume of viral vectors lower than 100µl: just before cells transduction, remove the viral vectors from the -80°C freezer and thaw them on ice at 4°C. 5min before transduction take the viral vectors off the ice and allow them to warm up at room temperature.
Our lentiviral vectors are packaged in working aliquots. Minimize freezing and thawing; a 15-20% reduction in titer for every freeze-thaw cycle should be expected. If aliquoting is necessary, please keep all samples on ice.
We do not recommend using the viral vectors more than once.
The optimal media will vary based on the somatic cell type. Generally, the best medium to use is the medium you usually use in culture for your specific somatic cells. You don't need any specific medium related to lentiviral vectors.
Questions about fluorescent lentiviral vectors
These ready-to-use, high-titer lentiviral particles contain a gene that encodes for a fluorescent protein—five fluorescent protein options are available. The fluorescent proteins are not targeted to a specific location in the cells so will be present mainly in the cytoplasm of the cell.
Click on Clontech's fluorescent protein for detailed information on excitation and emission spectra, recommended filter sets, antibodies and other applications of fluorescent proteins from Clontech.
Click on Evrogen's fluorescent protein for detailed information on excitation and emission spectra, recommended filter sets, and antibodies and other applications of fluorescent proteins from Evrogen.
Each subcellular localization vector expresses a bright fluorescent protein fused to a domain that targets it to a specific cellular compartment. Click on subcellular fluorescent lentiviral vectors for more information on available lentiviral particles for subcellular localization studies.
Questions about reprogramming lentiviral vectors
The lentiviral vectors are provided in either three 20µl aliquots or in one single 20 µl aliquot, both at a concentration greater than 1x109 TU/ml.
The titer of all our standard lentiviral vectors products is > 1x10E9 TU/ml. The exact titer can vary for each lot. Please refer to the Certificate of Analysis for the titer of a particular lot.
These vectors are high titer, purified, ready-to-use, recombinant lentiviral vectors that can be used to reprogram somatic cells into induced pluripotent stem (iPS) cells. These lentiviral vectors contain genes for the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), expressed either individually or as a polycistronic transcript. The expression of these transcription factors in somatic cells has been shown to successfully generate iPS cells (1).
Using an M.O.I. of 5, one set (3 x 20 µl) of polycistronic lentiviral vectors at 1x109 TU/ml can be used to reprogram >1x107 cells.
The Flash Therapeutics lentiviral vectors has a reprogramming efficiency between 0.01% to 1%; a 100-fold increase in efficiency over standard methods used to generate IPS cells.
Colonies will begin to form approximately 2 weeks post-transduction. Select iPS colonies 20-25 days after transduction.
- Rashid S. T, Corbineau S, Hannan N, Marciniak S.J, Miranda E, Alexander G, Huang-Doran I, Griffin J, Ahrlund-Richter L, Skepper J, Semple R, Weber A, Lomas D. A, Vallier L. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest., 2010, 120 (9), 3127-3136.
- Lapillonne H, Kobari L, Mazurier C, Tropel P, Giarratana M-C, Zanella-Cleon I, Kiger L, Wattenhofer-Donze M, Puccio H, Hebert N, Francina A, Andreu G, Viville S, and Douay L. Red blood cells generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica. 2010, 95 (10), 1651-1659.
- Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev., 2009, 23: 2134-2139.
- Vallier L, Touboul T, Brown S, Cho C, Bilican B, Alexander M, Cedervall J, Chandran S, Ährlund-Richter L, Weber A, Pedersen R.A. Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells. Stem cells, 2009, 27 (11) : 2655-2666.
Visit the Flash Therapeutics website for more product information, data, protocols and troubleshooting tips. For any product or service inquiries, feel free to contact us.