About the virus vectors

What are retro (RV) or lentiviruses vectors (LV) ?

 

Retroviral vectors (RV) and Lentiviral vectors (LV) are derived respectively from gammaretroviruses and Lentiviruses. These two viruses belongs to the same family:Retroviridae family(class 6). RV and LV are able to "transduce" many cell types. With the help of this RV or LV, your DNA sequence of interest is inserted into host genome allowing to generate stable cell lines. Delivery of genes into cells by a virus is termed transduction and the cells are described as transduced.

 

Our LV vector derived from lentivirus HIV-1 and our RV from Murine leukemia virus (MuLV).

 

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Why use LV or RV ?

LV have the capacity to efficiently transduce many cell type including non-dividing cells. However, RV are able to transduce only cell in division. LV and RV are able to maintain stable long-term transgene expression due to their capacity to integrate their genome into host target cells.  RV and LV exhibit a relatively large capacity of cDNA cloning close to 8 kb. The final integrated sequence that encodes for the gene of interest do not encode for any viral protein derived from the packaging parental virus.                    

RV and LV can be useful for the following applications:

• Gene delivery into hard-to-transfected cell types (such as neuron cells), primary or drug-arrested cells: mainly for LV.

• Integration of your specific gene sequence into adherent and suspension cell DNA  

• Highly reproducible gene delivery

• In vivo imaging: dual expression of your target gene and a reporter gene (i.e. luciferase or EGFP).

 

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How are our LV produced ?

Lentiviral vectors are produced by transient transfection of HEK293 cell lines by plasmids carrying packaging functions and the recombinant construct with the cDNA or shRNA sequence of interest. To produce a lentiviral particle, several packaging functions are necessary:

• GAG + POL: the enzymatic products of the pol gene and the structural proteins encoded by the gag and env genes lead to the budding at the plasma membrane of the producer cell line. After packaging, all these functions will contribute to the efficiency of the early steps of viral cycle: cell entry, reverse transcription, nuclear translocation and integration.  Here, the parental or viral replicative cycle has finished and the following events are only transcription and translation of foreign inserts without any viral proteins synthesis.  

• REV: Only for lentiviruses, a virally-encoded post-transcriptional activator called Rev is necessary for efficient gene expression.    

• Glycoprotein: The parental envelope protein can be substituted by the corresponding protein of another virus to modify the tropism of the viral vector. The G protein of vesicular stomatisis virus (VSV-G) is classically used to pseudotype lentiviral.

In order to produce lentiviral vectors, HEK293 cells are co-transfected with:

• the gene transfert vector: a plasmid containg your gene of interest (GI) with a suitable promoter (P), a selection maker if needed and the packaging HIV-1 sequence (Psi) between the LTRs

• the auxilary plamids containing HIV-1 GAG and POL genes

• the enveloppe plasmid: VSV-G or other viral envelopes.

Then the supernatant, containing LV, is collected during 3 days and used to transduced your cells lines.

 

Viral supernatant is directly harvested (approx. titer = 10E6 TU/ml) or can by concentrated by ultracentrifugation (100-200 x; approx. titer = 10E8 TU/ml).

The GIGA Viral vector plateform provides you with different batches of LV:

The new generated cell lines are tested for RCL (Replication competent Lentiviruses).

 

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How are our RV produced ?

Retroviral vectors can be produced by transient transfection of packaging cell lines.  These cell lines allow the generation of helper free ecotropic and amphotropic retroviruses.The Gene transfert vector containing the gene of interest (expression driven by LTR promoter) and the packaging signal (Psi) is transfected into phoenix cell. The surpernatant is the harvested and used to transduce your cell lines.

 

 Viral supernatant is directly harvested (approx. titer = 10E6 TU/ml) or can by concentrated by ultracentrifugation (100-200 x; approx. titer = 10E8 TU/ml).

 

The GIGA Viral vector plateform provides you : 300 μl of concentrated lentiviral vectors at a titer no less than 1E+08 TU/ml (upon request: > 1E+09 TU/ml).

The new generated cell lines are tested for RCR (Replication competent Retroviruses).

 

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What is the genome structure of HIV-1 ?

HIV-1 genome organization is represented below.

The plasmid containing the Gene transfert vector contains:

• the two LTR (with a mutation in the U3 region in order to obtain a self-inactivating (SIN) vector

• the packaging signal

• a promoter for the gene of interest

• the gene of interest (or shRNA 

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What are RCR and RCL ?

 

RCR= Replication Competent Retrovirus

RCL= Replication Competent Lentivirus

How  does this recombination occur ?

Recombination between

• Packaging and gene transfer vectors
• Retroviral vector and cellular DNA (endogenous viral sequence: murin cells)
• Retro/lentivirus vector and other retroviruses

 

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What are the Methods for RCR/RCL detection ?