Frequently Asked Questions

Frequently Asked Questions on Lentiviral vectors and LentiFlash® particles

GMP dyptique

General Questions

How are Flash Therapeutic lentiviral vectors different from other lentiviral vectors?

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.

How is the titer (TU/ml) of the lentiviral vectors determined?

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.

What is the titer of my lentiviral vectors?

The lentiviral vectors titer varies for each lot. Please refer to the certificate of Analysis (CoA) for the titer of your particular lot.

How many lentiviral particles should I add to my cells?

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

What are the safety concerns about the use of lentiviral and retroviral vectors?

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.

How should the lentiviral vectors be stored?

Upon receipt, lentiviral vectors must be stored at -80°C.

How should lentiviral vectors be thawed?

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.
Can the lentiviral vectors be thawed and refrozen?

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.

Can the lentiviral particles be used more than once?

We do not recommend using the viral vectors more than once.

What type of medium should be used during transduction of my somatic cells?

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

What are 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.

Where can I learn more about the fluorescent proteins used in the subcellular localization and marker lentiviral vectors?

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.

How do the subcellular localization lentiviral vectors work?

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

How are the lentiviral vectors products provided by Clontech supplied?

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.

What are the titers of the lentiviral vectors?

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.

What are Flash Therapeutics reprogramming lentiviral vectors?

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).

How many cells can be reprogrammed with one set of polycistronic lentiviral vectors?

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.

What reprogramming efficiency should be expected with these products?

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.

How long will it take for iPS colonies to form after transduction?

Colonies will begin to form approximately 2 weeks post-transduction. Select iPS colonies 20-25 days after transduction.

Are there any publications that reference use of the reprogramming lentiviral vectors?
  1. 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.
  2. 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.
  3. 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.
  4. 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.
Where can I find more information?

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.