Depends on how accurate you will want to titer the vector. Without having a fluorescent marker gene you will have to use a qPCR based approach to get vector genome / ml titer. If you have LV with a GFP marker you can determine transducing units/ml and also run on qPCR along with your shRNA vector to estimate transducing units on your cell of interest. If you do this make sure that both vectors are pseudotyped with the same envelope

As an alternative can use p24 ELISA to estimate vector particles.

Good luck

Hi Virginie,

You can titrate lentiviruses by:

1.Real time PCR by designing primers specific to lentiviral genes present in your construct for regions like gag, LTR etc.

2.By p24 Elisa if you are using HIV ShRNA construct.

or by commercial protocols and links below. Also find the attached pdf from Sigma

http://www.clontech.com/US/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Titration/qRT-PCR?sitex=10020:22372:US

Some papers with pubmed ID

PMID:17510916

PMID:16836756

All the best

Manoj

Hi Virginie,

You can titrate lentiviruses by:

1.Real time PCR by designing primers specific to lentiviral genes present in your construct for regions like gag, LTR etc.

2.By p24 Elisa if you are using HIV ShRNA construct.

or by commercial protocols and links below. Also find the attached pdf from Sigma

http://www.clontech.com/US/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Titration/qRT-PCR?sitex=10020:22372:US

Some papers with pubmed ID

PMID:17510916

PMID:16836756

All the best

Manoj

Hi Virginie,

You can titrate lentiviruses by:

1.Real time PCR by designing primers specific to lentiviral genes present in your construct for regions like gag, LTR etc.

2.By p24 Elisa if you are using HIV ShRNA construct.

or by commercial protocols and links below. Also find the attached pdf from Sigma

http://www.clontech.com/US/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Titration/qRT-PCR?sitex=10020:22372:US

Some papers with pubmed ID

PMID:17510916

PMID:16836756

All the best

Manoj

The sigma pdf

Hi Virginie. You can use qPCR for LV titration. There are many protocols available. We routinely employ the one published by Scherr et al. (BioTechniques,2001, 31:520-526) to titer ours (total LV per ul). It was described that 1% of total LV are TUs, which is true in our hands. You can find this pub in the protocol section of our website (www.rodriguezlab.com). Good luck! Cheers.

Hi Virginie

This is all you need to know (http://www.ncbi.nlm.nih.gov/pubmed/19300443). There are plenty protocols for qPCR titre determination of lenti - however the majority of these fall far short of the mark (ie they measure genome copies in a tube of 'purified virus'). What they don't generally take into account is the inefficiency of the packaging process (especially in the case of gene-modified lenti vectors (as opposed to wild type HIV)). This isn't restricted to lenti and applies to most (if not all) viral vectors used in gene therapeutic approaches. I'd strongly recommend the protocol in the attached paper (see 19.A) which requires the viral genome to be efficiently packaged, a virion produced, and that virion to infect a target cell (which is what you ultimiately want) and then assesses viral copy number per cell. It's the next best thing to flow cytometric analysis of cells infected with fluorescent protein expressing lenti's.

Good luck

al

Thanks a lot everybody for all these tips and information :)

An alternative method to those described above is to determine your viral titer by infecting a target cell line, for example HEK 293 cells, followed by antibiotic selection. An advantage of this method of titration is that you will obtain the titer of intact infectious particles, whereas determination of viral genes by qPCR may overestimate your titer, particularly in case of low quality viral preparations. To be able to perform such experiments your lentiviral vector has to harbor an antibiotic resistance gene. Pseudotyping with VSV-G during viral packaging ensures that you will be able to infect a large panel of cells due to its broad cell tropism.

Here is a paper giving the pros and cons of each method.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1534021/

You could try using the p24 assay if you are looking for the titer prior to transducing, particularly if you have a specific MOI that you would like to use.  It's quick and easy to do as it is an ELISA assay.  I've used this routinely to titer my virus.  Here is a link for the one that I use.  For this kit, I make a 1 x10-6 dilution (in water) of concentrated virus (following 18 hour centrifugation) prior to adding the lysis buffer.

http://www.zeptometrix.com/store/hiv-1-p24-antigen-elisa-1x-96-well/ 

Let me know if you need any help with the protocol using this method.

Alternatively,  If you want to check the level of transduction within your target cells, you can perform qPCR.  http://www.clontech.com/US/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Titration/qRT-PCR

Hope that helps

Determining the concentration of lentiviral particles is crucial for quantifying the amount of virus to be used in experiments. There are several methods to measure lentiviral titers, each with its own advantages and limitations:

Each method has its own advantages and is suitable for different purposes. For instance, p24 ELISA and reverse transcriptase assays are quick and suitable for estimating the overall viral particle concentration, while functional titering methods like the infectious unit assay or qPCR-based assays provide a more accurate measure of the number of infectious particles, which is crucial for experiments where precise dosing is needed. The choice of method often depends on the resources available, the level of accuracy required, and the specific application of the lentivirus.

l With this protocol list, we might find more ways to solve this problem.