Frequently asked questions about lentiviral particles

Flash Therapeutics has developed a proprietary purification platform for viral particle production. Purification levels are tailored to the target-cell type. Flash Therapeutics has developed a specific viral particle purification level for transduction of primary somatic cells to generate high-titer, highly pure viral particles that do not affect cell viability and proliferation.

The particles are provided in 20-µl aliquots, at a concentration greater than 1 x 10⁹ TU/ml.

The titers of all lentiviral particle products provided by Takara Bio are >1 x 10⁹ TU/ml. The exact titer varies by lot; refer to the Certificate of Analysis for the titer of a particular lot.

Lentivirus titer can be measured a number of ways; some vendors measure the total number of particles in a 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 1 x 10⁵ HCT116 cells with several serial dilutions of viral supernatant.

Because the titer of each lot of particles varies, the volume required for transduction will also vary. The titer can be found on the Certificate of Analysis provided with each viral vector. Use the formula below to calculate the volume of viral vector for transduction. Refer to the product user manual for additional information.

The optimal MOI will vary, and a range of MOIs should be tested when transducing a lentiviral construct into a cell line for the first time. Use an MOI between 10 and 100 as a starting point. Refer to the link below for transduction examples for a variety of cell lines and cell types. Recommended cell seeding density and M.O.I. for various cell types »

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 particle products provided by Takara Bio are self-inactivating (SIN). Thus, the viral particles maintain the ability to infect a wide range of cell types, but they are not capable of producing replicative particles from transduced cells as assessed by RCL/RCR testing. Refer to the MSDS documents for more information.

Upon receipt, store at –80°C.

Just before use, remove the particles from the –80°C freezer and thaw on ice (4°C). Five minutes before transduction, the vial should be removed from ice and allowed to equilibrate to room temperature. Once thawed, the vectors should be used for transduction as soon as possible to avoid degradation.

To prevent heat shock, allow culture medium to equilibrate to room temperature fully before diluting vectors and cells.

The viral particles 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, keep all samples on ice.

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

The purification level of Flash Therapeutics lentiviral particles distributed by Takara Bio allows for in vivo use, such as local injection of viral particles and engraftment of genetically modified primary cells. The lentiviral particles can be injected in all tissue types depending on the application, including dentate gyrus, subventricular zone, muscle, and blood, to achieve high transduction efficiency. Hematopoietic cells, lymphocytes, and tumor cells can be also transduced without inducing cytotoxicity, even at a high multiplicity of infection (MOI). The cells can then be re-engrafted into animals to develop genetically modified animal models. If you need to perform transgenesis experiments or preclinical trial studies for gene therapy purposes, we recommend using lentiviral particles produced with a specific purification process that includes a supplementary chromatography step. Please contact Flash Therapeutics at project@flashtherapeutics.com if you need further details about in vivo-quality, custom-made lentiviral vectors.

Click here for detailed information on excitation and emission spectra, recommended filter sets, and antibodies and other applications of Takara Bio's fluorescent proteins.

Each vector expresses a bright fluorescent protein fused to a domain that targets it to a specific cellular compartment. Click here for more information on available lentiviral particles for subcellular localization studies.

These ready-made, high-titer lentiviral particles contain a gene that encodes a fluorescent protein—five fluorescent protein options are available. The fluorescent proteins are not targeted to a specific location in the cells so they will be present mainly in the cytoplasm. Using these lentivirus particles, cells can be stained red, green, cyan, or yellow.