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Viral Transduction


Products >  Viral_Transduction >  Lentiviral_Particles >  lentiviral_particles_overview

High-Titer, High-Purity Lentivirus Particles

Clontech has partnered with Vectalys to provide ready-made lentiviral particles for use in tet-inducible expression studies, to create induced pluripotent stem (iPS) cells, and for fluorescent marker studies. We believe these are the highest-quality ready-made lentiviral particles available, due to Vectalys’ unique method of producing high-purity, high-titer particles with superior performance.

What you can expect with lentiviral particles from Vectalys:

  • 100% transduction of many primary cells (including neurons, endothelial cells, and stem cells)
  • High expression in primary cells when delivered at a high MOI
  • No cytotoxic effects
  • Functional titer—not just a particle count

What is different about lentiviral particles produced by Vectalys and sold by Clontech?

Unlike ready-to-use lentiviral particles from other suppliers, the particles in our catalog are produced via a multi-step purification process which results in very high titers and purity. The high titer increases transduction efficiency, and the high purity reduces cytotoxicity, so you can successfully transduce a large number of different cell types—including primary, immortalized, adherent, and suspension cells—and retain high cell viability.

State-of-the-art lentivirus production platform

Vectalys has developed a unique process for the production of high-titer viral supernatant. This process involves generation of virus in 293T cells in a serum-free medium, followed by back-to-back tangential ultrafiltration with diafiltration to concentrate and purify the particles. Finally, after titration and assaying for RCL, the safe, high-titer, high-purity lentiviral vectors are ready for transduction of your cells.

Process for generating high-titer lentiviral particles

Overview of the process for generating high-titer lentiviral particles. Through this proprietary, large-scale process Vectalys is able to produce very high-titer, high-purity lentiviruses ready for transduction of many different cell types.

We supply a functional titer, not simply a particle count

Not all advertised titers are the same! Most vendors of ready-made viral particles determine their titers using a simple particle count. This count will include free capsid protein, nonfunctional and immature virions, as well as functional virus.

For every lot of our lentiviral particles, we provide a functional titer of >1 x 109 transduction units per ml (TU/ml). The functional titer is not just the total particle number, it is a measurement of the total number of virions capable of integrating into cells. Compared to the titration methods of other vendors, Vectalys’ method ensures an accurate count of functional particles. In real terms this means our viral preps contain up to 100-fold more infectious particles than preps from other vendors.

Transduction efficiency of GFP-expressing lentiviral particles

Transduction efficiency of a Vectalys GFP-expressing lentiviral vector in IMR90 cells. Even with a low MOI of 5, >95% of cells are transduced using this high-titer lentivirus. Very high expression can be achieved by increasing the MOI.

Why are titer and purity important?

Higher titers increase the speed, ease and efficiency of lentiviral transductions. Higher lentiviral titers allow you efficiently infect even hard-to-transduce cell types at a lower MOI. The high purity of the lentiviral particles dramatically reduces cytotoxicity further increasing the efficiency of transduction of delicate cells.

Lentivirus particle purity

Comparison of the purity of lentiviruses generated with Vectalys’ method or a classic purification method. The high purity of Vectalys lentiviral particles is clear when comparing the two methods; even when Vectalys particles are loaded at a 10-fold higher amount, very little contaminating protein is seen.

Why serum-free medium?

Production in serum-free medium greatly improves the quality of the lentivirus as a vector for gene delivery: eliminating serum from the lentiviral supernatant reduces the levels of contaminating proteins that need to be removed in the purification process. This allows for increased titer (TU/ml), decreased physical particles to transducing units ratio (PP/TU; more effective particles than ineffective particles), and increased transduction efficiency of the virus prep. The Vectalys particles display higher purity than those concentrated by classic ultracentrifugation methods—even when the Vectalys particles are loaded at a 10-fold higher concentration.

Products available from Clontech

Viral Particles for Tet-Inducible Gene Expression

Clontech offers ready-made lentiviral particles to introduce constitutive expression of the Tet-On 3G transactivator in your cells of choice. In the presence of doxycycline (a tetracycline derivative), the Tet-On 3G transactivator binds to, and activates transcription of, the PTRE3G promoter. This allows you to precisely control transcription of any gene cloned downstream of PTRE3G. Learn more >>

Viral Particles for Subcellular Localization Studies

Constitutive Lentiviral Reporter Vectors are prepackaged lentiviral particles that deliver fluorescent proteins targeted to specific organelles or subcellular structures. These particles can be used to create reporter cell lines for the study of cytoskeletal and organelle structure and function in living cells or protein localization studies. Learn more >>

Induced Pluripotent Stem (iPS) Cell Production Systems

We also offer tet-inducible, polycistronic recombinant lentiviral particles for generating iPS cells. These particles encode the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) expressed from the PTRE3G promoter. Expression of the Yamanaka transcription factors in somatic cells has been shown to successfully generate iPS cells1. This polycistronic lentiviral vector is offered in a constitutive expression format as well. Finally, we have lentiviral particles constitutively expressing each Yamanaka transcription factor individually. Learn more >>

Schematic maps of expression plasmids used for preparation prepackaged, high concentration lentivirus particles (click to view larger versions).

Reference:

  1. Takahashi, K. et al. (2007) Cell. 131(5):861–872.

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