Lentiviral Particles from Vectalys
How are these viral particles different from other viral vectors?
Vectalys has developed a proprietary purification platform for viral particle production. Purification levels are tailored to the target cell type. Vectalys 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.
How are the recombinant lentiviral particles supplied?
The particles are provided in 20-µl aliquots, at a concentration greater than 1x109 TU/ml.
What are the titers of the viral particles?
The titers of all lentiviral particle products provided by Clontech are >1x109 TU/ml. The exact titer varies by lot; refer to the Certificate of Analysis for the titer of a particular lot.
How is the titer (TU/ml) of the viral particles determined?
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 Vectalys 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.
How much of the viral particles should be added to my cells?
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.
|Volume of viral vector required (µl) =||Number of cells seeded||x M.O.I. x 1,000|
|Viral vector titer (TU/ml)|
What MOI should be used?
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 >>
What are the safety concerns for 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 Vectalys-manufactured lentiviral particle products provided by Clontech 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.
How should the viral particles be stored?
Upon receipt, store at –80°C.
How should viral particles be thawed?
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 avoid heat shock, allow culture medium to fully equilibrate to room temperature before diluting vectors and cells.
Can the viral particles be thawed and refrozen?
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.
Can the viral particles be used more than once?
We do not recommend using the viral particles more than once.
Can the viral particles be used in vivo?
The purification level of Vectalys lentiviral particles distributed by Clontech 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, incuding 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 Vectalys at firstname.lastname@example.org if you need further details on in vivo quality, custom made lentiviral vectors.
Questions about reprogramming lentiviral particles
What are reprogramming lentiviral particles?
These vectors are high titer, purified, ready-to-use, recombinant lentiviral particles that can be used to reprogram somatic cells into induced pluripotent stem (iPS) cells. These recombinant lentiviral particles 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).
- Takahashi, K., et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872
How many cells can be reprogrammed with one set of polycistronic viral particles?
Using an MOI of 5, one set (3 x 20 µl) of polycistronic viral vectors at 1x109 TU/ml can be used to reprogram >1x107 cells.
What reprogramming efficiency should be expected with these products?
The reprogramming lentiviral particles provide reprogramming efficiencies between 0.01% and 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.
What type of medium should be used for culturing somatic cells?
The optimal media will vary based on the somatic cell type. Generally, the best medium to use is the medium you use for your specific somatic cells.
Are there any publications that reference use of the reprogramming lentiviral particles?
- Rashid, S. T., et al. (2010) Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J. Clin. Invest. 120(9):3127–3136.
- Lapillonne, H., et al. (2010) Red blood cell generation from human induced pluripotent stem cells: Perspectives for transfusion medicine. Haematologica 95(10):1651–1659.
- Banito, A., et al. (2009) Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23:2134–2139.
- Vallier, L., et al. (2009) Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells. Stem Cells 27(11):2655–2666.
Questions about fluorescent subcellular marker particles
Where can I learn more about the fluorescent proteins used in the subcellular localization lentiviruses and marker lentiviruses?
Click here for detailed information on excitation and emission spectra, recommended filter sets, and antibodies and other applications of Clontech’s fluorescent proteins
How do the subcellular localization vectors work?
Each subcellular localization 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.
What are the fluorescent protein marker particles?
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 will be present mainly in the cytoplasm of the cell. Using these lentivirus particles, cells can be stained red, green, cyan, or yellow.
Available Ready-to-use Lentiviral Particles
|Reprogramming Lentiviral Particles||Generate iPS cells from somatic cells|
|Lentiviral Particles Expressing Fluorescent Proteins||Express fluorescent proteins in the cytoplasm for cell tracing and cell tracking experiments|
|Express fluorescent proteins in specific subcellular locations|
|Tet-On 3G Lentiviral Particles||Tet-inducible gene expression studies|