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VECTOR OPTIONS

 

 

  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.

 

Short Code
Full Name
Origin
Cell Specificity
AP2
Fragment enhancer + adipocyte P2 minimal
Mouse
Adipocytes
ARR3lg
Arrestin 3 long
Pig
Retina
ARR3sh
Arrestin 3 short
Pig
Retina
CAG
Cytomegalovirus early enhancer + chicken b-actin promoter
Synthetic
Ubiquitous
CaMKII
Ca2+/calmodulin-dependent protein kinase II
Mouse
Neurons
CMV
Cytomegalovirus promoter
Cytomegalovirus
Ubiquitous
EF1a
Elongation factor 1 alpha 1
Human
Various organs
FLT1
Fms-like tyrosine kinase-1
Human
Endothelial cells
GFAP
Glial fibrillary acidic protein
Human
Glial cells
NSE
Neuron specific enolase
Rat
Neurons
PGK
Phosphoglycerate kinase 1 promoter
Mouse
Ubiquitous
RHO
Rhodopsin
Mouse
Retina
SYN
Synapsin
Human
Neurons
UBI
Ubiquitin C
Human
Ubiquitous

  How do I choose between the various integrases?

The first thing to determine to be able to choose among the integrase is to define what kind of kinetics of expression you need for your experiment. From there you can choose the right mutation as follows:

 For a long-term and high expression in dividing and non-dividing cells:

  • IN(WT) is the wild type integrase. It will be the perfect choice if you don't have particular needs in terms of integration
  • IN(R166A) and IN(Q168A) are LEDGF mutants. Usually, the vector genome integration in the host cell chromatin is subject to a bias toward transcriptionally active regions due to an interaction between the integrase and LEDGF, a cellular factor. These two mutations are expected to abolish Integrase–LEDGF interactions and make the vector genome integration profile more random in the target cell genome.

 For a short-term expression in dividing cells (or long-term in non-dividing cells):

IN(D64V), IN(N) and IN(LQ) are three mutations that disable the integration capabilities of the vector. These three mutations exhibit different profiles of expression, a particular behavior in terms of residual integration frequency and level of transgene expression. IN(D64V) shows the highest level of expression and the highest residual integration frequency; IN(N) shows the lowest level of expression and the lowest residual integration frequency; IN(LQ) shows 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.

 

 For a transient expression:

RT(D110E), RT(E478Q) and RT(D1003/E478Q) are three mutations that disable the reverse-transcriptase of the vector, making the expression highly transient. These three mutations exhibit different profiles of expression, depending on your experimental context. We recommand that you test them to determine which one is the best suited for your purposes.

 

 
Name
Integrative
Mutation
Effect
Integrase Mutants
IN (WT)
Yes
None
Strong and robust expression through integration in the host cell chromatin.
IN (R166A)
Yes
Substitution in the codon 166 (166 R>A)
IN (Q168A)
Yes
Substitution in the codon 168 (168 Q>A)
Abolished Integrase-LEDGF interaction. Vector genome integration profile is no longer biased toward transcriptionally active regions.
IN (D64V)
No
Substitution in the catalytic site (64 D>V)
IN (LQ)
No
Substitution in the LQ basic region (186 K>Q, 214 Q>L, 216 Q>L)
IN (N)
No
Substitution in the N basic region (262 AAH>RRK)

Deficient for integration. The transgene expression relies on the episomal forms of the vector genome.

  • Long-term expression in non-dividing cells
  • Short-term expression in dividing cells
Reverse-transcriptase Mutants
RT(D110E)
No
Substitution in the polymerase active site (110 D>E)
RT (E478Q)
No
Substitution in the RNase H active site (478 E>Q)
RT (D110E/E478Q)
No
Two substitutions (110 D>E / 478 E>Q)

Deficient for reverse transcription. There is no DNA synthesis and the RNA vector genome is handled as a mRNA by the host cell.

  • Short-term expression in non-dividing cells
  • Short-term expression in dividing cells

 

  How do I choose the best envelope?

 

 

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 envelope 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 suits you best.

  • VSV is the envelope glycoprotein from the vesicular stomatitis virus. Lentiviral vectors produced with this envelope are expected to have a broad tropism and transduce a wide range of cell types.
  • Mokola is the envelope glycoprotein from the Lyssavirus Mokola. Lentiviral vectors produced with this envelope are expected to be glial cell specific in vivo.

  Which titer should I order?

Lentiviral vectors are available at two levels of concentration:

  • For integrative vectors (IN(WT), IN (R166A), IN (Q168A)), the high titer is 108 TU/ml, and the very high titer is 109 TU/ml
  • For non-integrative vectors (integrase mutants and reverse-transcriptase mutants), the high titer is 107 TU/ml, and the very high titer is 108 TU/ml. For these vectors, titers are a bit lower because of their particular kinetics of 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 the TU titer has been determined by serial dilution on 293T cells.

You can order vectors by 50 or 200 μl. If you wish to order a specific volume or titer, it can easily be done through our On-Demand service. Don't hesitate to contact us.