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VECTOR TITERS

 

  How are GEG Tech vectors titered?

GEG Tech produces each batch of vector with great caution, routinely established protocols and regular quality controls all along the production process. The final step consists in determining the concentration of the produced vector. The concentration is determined through 2 parameters:

  • the RNA content
  • the Transducing Unit content (equivalent)

  What is RNA titer?

Each vector particle contains 2 molecules of RNA genome. For each vector production, the concentration of RNA molecules is determined per microliter based on an optimized protocol of RNA extraction, reverse transcription and real time PCR. As RNA genomes are rapidly degraded when not protected into the vector capsid, the RNA content gives a relevant measurement of the vector efficiency, in contrast to the widely used p24 content, as p24, the capsid protein, can be either free or associated to a particle containing no RNA genome.

 

  What is the TU equivalent?

GEG Tech vectors are available at High Titer (107 TU/mL for Lenti-ONE™ Epi and Lenti-ONE™ Trans and 108 TU/mL for Lenti-ONE™) and High Titer+ (108 TU/mL for Lenti-ONE™ Epi and Lenti-ONE™ Trans and 109 TU/mL for Lenti-ONE™).

The transduction efficiency of a vector is the result of the particular combination of:

  • the vector (its envelope, its integrase, its PBS, its promoter, its transgene, the presence of a post transcriptional regulatory element…)
  • the target cells (their cell type, their progression in the cell cycle, their density…)
  • the analytical method used to quantify the transgene expression

To allow you to compare vectors among each other, despite their different envelopes, promoters, transgenes and other features, GEG Tech provides for each vector production a TU equivalent titer, which is based on the RNA content and compared with a standard vector expressing GFP under the transcriptional control of a CMV promoter for which TU titer has been determined by serial dilution on 293T cells.

 

  How do I determine the quantity of viral vectors to add to my cells?

The transduction efficiency of a given vector (with a particular envelope, promoter, transgene…) depends on the target cells and its adequacy with said target cells. Consequently, the optimal transduction conditions should be optimized for your particular experimental context, especially for each new cell type you are assaying.