Giulia, and do you have some marker gene in your plasmid with the gene of interest (for example, antibiotic resistance gene)? You can use it in cheap titer determination by formation of the resistant colonies.

Also, there are some commersially available kits for titer determination, based on p24 level assay.

The problem is that lentiviral titer varies greatly in defferent rounds of production. During in vivo experiments infections with different MOI may weakly affect gene of interest (so, you would not notice any changes in the western of animal tissue samples), but may affect, for example, cells' viability. So, it is beter to know the MOI of your preparation (and the reviewers will undoubtedly ask this question during the publication process).

Hi, Giulia.

For lentiviral transduction in vivo you need to know the titer of your lentivirus to dilute it the way you should dilute it to get multiplicity of infection, appropriate for your experiment. So, the first step will be determining the titer of lentivirus, then you need transduce it to your cells at the appropriate MOI and then - check the change in expression by PCR or Western Blot. Also I advice you to check the expression change after your in vivo experiment by taking samples of target tissue from the experimental animals and subjecting them to PCR or Western.

If your virus express GFP - it will be much easier to determine the titer by preparing a range of lentiviral preparation dilution and transducing them to the cells, seeded in the same density. And simply by observing the cells under fluorescent microscope you will be able to count the titer.

I agree with Darya Meshalkina, the methodologies PCR and WB will only answer the physical nature of the produced lentivirus. The functional titer (infectivity) which is primarily important for in vivo, will only be determined by transduction assay.

Thanks for the very clear replies,

I will indeed also perform an experiment on the target tissues to check if it worked. Unfortunately, my control plasmid is a different one, so I will have the GFP plasmid and the plasmid with the gene of interest, but no GFP-gene of interest together...(it would have been too easy).

But that's already good to have some answers,

thanks guys!

Giulia, and do you have some marker gene in your plasmid with the gene of interest (for example, antibiotic resistance gene)? You can use it in cheap titer determination by formation of the resistant colonies.

Also, there are some commersially available kits for titer determination, based on p24 level assay.

The problem is that lentiviral titer varies greatly in defferent rounds of production. During in vivo experiments infections with different MOI may weakly affect gene of interest (so, you would not notice any changes in the western of animal tissue samples), but may affect, for example, cells' viability. So, it is beter to know the MOI of your preparation (and the reviewers will undoubtedly ask this question during the publication process).

There is an antibiotic resistance gene, for hygromycin, so that is something I can do of course.

Besides this, I am working with something that somebody else has made, so I will also need to send my plasmids for sequencing, and then I will also start transfecting my cells to stimulate then afterwards to check the amount of my gene of interest.

But I will for sure detect the MOI of my preparation. Because I think I may regret if I don't.

Thank you guys, With this discussion, I know many more ,,,, By the way, this time is firstly from me

You can prepare lentiviral system for luciferase gene and use it as a control for your system. Luciferase activity is quite easy to measure and much more quantitative than western blotting. With this way you can optimize your condition if you want more less expression in target cells.

Also you can add different luciferase (renilla or firefly) plasmid during transfection and see transfection efficiency.

Since these viruses are not replication competent there isn't many way to see how many virus you have.

There is a Lentivirus Webinar coming up that may able to help address your concern and any additional questions.

Title: Knockdown, Knockout, Validate: Lentivirus delivers payload in vitro and in vivo Date: Wednesday, May 16, 2018 Duration: 60 minutes Times: 10:00 AM (CST) Speaker: @Christy Hoffmann

What you will learn: Lentivirus biosafety features and best handling practices Flexible customization options to fit your needs Techniques to incorporate lentivirus into your gene studies Insight from a leading Lenti manufacturing expert

Register here: https://www.sigmaaldrich.com/life-science/learning-center/customer-education/lentivirus.html