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VECTOR DESIGN

 

  How do I choose between vector lines: Lenti-ONE™, Lenti-ONE™ Epi,

Lenti-ONE™ Trans?

 

 

Most transgenes are available as Lenti-ONE™, Lenti-ONE™ Epi or Trans. The choice among these 3 types of lentiviral vector depends on your purposes. Basically:

  • Lenti-ONE™ mediates high level of transgene expression and the expression is stable in dividing and non-dividing cells, due to integration of the vector genome into the host cell chromatin. The expression is driven by an internal promoter which can be chosen to drive controlled expression.
  • Lenti-ONE™ Epi mediates lower level of transgene expression and the expression is transient in dividing cells and stable in non-dividing cells, due to an integration deficiency. The expression is driven by an internal promoter which can be chosen to drive controlled expression.
  • Lenti-ONE™ Trans mediates fully transient and low level of transgene expression in dividing and non-dividing cells. It is particularly suited when long-term transgene expression is deleterious. The cell targeting can only be defined through the choice of the envelope as the vector genome contains no promoter.

 

  How do I choose the best envelope?

 

Video transcript:

Modulation of cell-specificity expression through the choice of vector envelope

The very first step of transduction is the fixation of the viral particle to the target cell. For that to happen, there must be a recognition between the envelope glycoprotein of the vector, and receptors on the cell membrane. The existing receptors on the cell membrane vary according to multiple factors, including the cell type. Some receptors are ubiquitous and some are specific to a cell type. GEG Tech offers two types of lentiviral vectors pseudotyped with two different envelope glycoproteins: VSV and Mokola. The VSV-G glycoprotein binds with a membrane receptor present on most cell types. Therefore vectors with a VSV pseudotype are able to transduce a wide range of cell types. The mokola-G glycoprotein, on the contrary, binds to a receptor specific to glial cells. Only glial cells will be transduced, and your transgene will have a cell type-specific expression.

Let’s have a visual on real cells with sections of murine brain after stereotactical injection of PGK GFP lentiviral vectors. With VSV pseudotyped vectors, you can see that both neurons and glial cells express GFP. But with Mokola pseudotyped vectors, while glial cells widely express GFP, no expression can be seen in neurons.

Choosing the suitable envelope allows to achieve cell type-specific expression.

 

The choice of the envelope is an important feature, especially for in vivo applications, because it affects the area of expression by determining the nature of the transduced cells. A pantropic envelop allows to transduce most cell types and widespread transgene expression; in contrast, a specific envelop targets transduction of specific cell types, depending on the preferred entry mechanism and the receptors located at the cell surface, transgene expression is thus restricted to a/some particular cell type(s). As each experimental context is particular, we advise you to test our different envelopes in your own system to determine which one suit you best.

 

  How do I choose the best promoter?

The choice of the promoter is an important feature because it determines in which cells the transgene is expressed and at what level. Some promoters drive ubiquitous or cell specific transgene expression and low, medium or high level of transgene expression. In addition, the strength of a promoter can be modulated by the nature of the target cells. As every experimental context is specific, we advise you to test different polymerase II promoters in your own system to determine which one will suit you best.

 

  How do I choose between the various integrases?

Lenti-ONE™ Epi are episomal vectors but display a residual integration activity that is inherent to the DNA vector genome. GEG Tech offers several types of Lenti-ONE™ Epi with different integrase mutations, each one showing a particular behavior in terms of residual integration frequency and level of transgene expression. Thus, Lenti-ONE™ Epi IN(D64V) show the highest level of expression and the highest residual integration frequency; Lenti-ONE™ Epi IN(N) show the lowest level of expression and the lowest residual integration frequency; Lenti-ONE™ Epi IN(LQ) show intermediate level of expression and residual integration frequency. As every experimental context is specific, we advise you to test different integrase mutations in your own system to determine which one will suit you best.