You can also try to enhance your transduction by spinning the cells with the lentiviral sup. Typically 800 x g at 32°C for half an hour works really well. Combination of polybrene at 8µg/ml with spinoculation gives the best results.

Hi, when u do transfection do u see formation of the syncytial cells? , It might be a problem of the viral production or the efficiency is not high enough, you can also try to use fresh viral supernatant instead of frozen one.

Hi,  by using RT-qPCR to titer lentiviral particles could be overestimated. Because some non-effective particles also contribute the number.

Two possible solutions:

1) Verify the titer of effective particles. E.g. Make serial dilutions of your supernatant and transduce K293T cells, check the GFP positive percentage.

2) Concentrate the virus by using ultracentrfugation. For neuroal transduction, I use MOI between 2.5 and 3 and get 80% positive cells.

A manuscript  of "Lentiviral vector production, titration, and transduction of primary neurons." was attached.

Hope it helps!

Good luck!

I agree with Baojin; qPCR titres can overestimate your titre, so perhaps you are not adding enough.  However, just to start from the beginning, were your viral producer cells expressing GFP?  That's a quick way to make sure you haven't made a mistake right at the beginning.

I hope you manage to solve the problem, good luck.  If all else fails, ask your collaborator for more concentrated vector!

I agree with the previous posts - it's good check your producer cells to rule out a problem with the genome plasmid, and the most likely reason for no GFP positive cells is too little vector.

When you said you have a concentrated stock that works, did you transduce at a similar MOI this time? If not you could easily confirm if particle number is the issue by transducing your cell line of interest with the same number of particles of the concentrated stock you know works.

I also agree that you should check the GFP expression of the cells after you transfect. Another factor that would be essential for making good virus is how you maintain your HEK293T cells. We usually never let them over grow and pass them either 1:5 or 1:10 for maitenance when they are at 80 - 90% confluence. For making virus, I would plate them at quite high density (1:3 or 1:4) so that they will be nearly confluence the next day. This makes sure that you have enough cell to produce virus but they are not clustered yet --> hence an optimal transfection efficiency.

If you need your virus for something like in vivo injection, I would recommend using concentrated virus. Check out this paper: 

"Optimized production and concentration of lentiviral vectors containing large inserts."

Hope this helps!

Hi All,

Thank you all , I do agree with all the points made,

1° I will use fresh virus rather than frozen.

2° I have calculated the MOI .

3° i WILL ADDITIONALLY USE ELISA.

4° HEK293T cells are seeded as mentioned, I have 80-90% confluence.

5° to make sure I have added enough , I have actually transduced 3 times still no GFP expression in my target cells.

Thank you all for your time and valuable inputs. 

Do you include polybrene in your transduction mixture?  It neutralizes charge repulsions between the virus and the cells and greatly enhances transduction efficiency.  2-10 micrograms per mL will probably be sufficient but check what is published for your cell line (toxicity issues) and the manufacturer's protocol.

You can also try to enhance your transduction by spinning the cells with the lentiviral sup. Typically 800 x g at 32°C for half an hour works really well. Combination of polybrene at 8µg/ml with spinoculation gives the best results.

Also, you probably just have not mentioned it in your question and are using envelope plasmid during transfection. However, if not, then you will have made particles that cannot enter the cell, but are still quantifiable by both RT-PCR and elisa.

Hi,

1. I`m quantifying cell death with the gene of my interest, so Im don`t use polybrene.

2. I do use plasmid encoding envelope for the production of virus, but I guess as you said, the viral particles are there but no infection.  

Hi,

In that case I would just stick to the tips already given here: check if the producer cells are expressing gfp, use the virus you collect to transduce HEK293T to rule out the MOI difference between your cells of interest and HEK293T. If you don't have an ultracentrifuge, you can use a compound called PEG-it, to precipitate your lentiviral particles and then reuspend them in a smaller volume to concentrate it. This way you can concentrate it up to 100 times. Good luck! 

Hi

First, Use the recommended ratio of plasmids for transfection. if your insert too heavy (more than 2 kb) use packaging plasmid 2 or even 3 times of usual.

Second, Do you check the transfection efficincy by GFP? Transfection lower than 80 % would not work as much in virus production.

Third, remove am equal volume of cell media and replace it with the same volum of media containing virus. For instance, try not to exceed the final media volume of the cells after adding the media containing virus more than 500 ul in 24-well plate.

Forth, keep in mind, before you do all above, check the accuracy of cloning and sub cloning process such as the direction of GFP, IRES, insert, ....

Have a good transduction

Mostafa

Did you first make sure your transfection worked. You can either check if your protein is expressing or you can look for GFP. 

This will atleast give you an  idea if your cells are transfected.

If they are transfected, they you might want to play around with your helper plasmids -their purity, concentration, ..

Hi,

The titer you mentionned by RT-qPCR seems low, generally we obtain 1Log more. Another thing is that lentiviral vectors are very sensible to the frozen when the prep is not concentrated, so I think you have two ways of improvement

- optimization of your production process

- conservation of your prep

If you can't to concentrate your prep I advise you to use it directly after production or to conserve it at 4°C, in this way you can conserve it during about one week.

Finally, if you don't manage to obtain the vectors that you want, our compagny offers this type of vector (CMV-GFP from 170E) and many more.

http://www.geg-tech.com/Vectors/index.php/line-lenti-one.html?env=14&integrase=19&promoter=35&wpre_=63

Best,

Nicolas

Nicolas

Just to add that Takara/Clontech offers complete solutions for Lentiviral Transductions.

Follow the link for easy protocols with required products to be more efficient.

http://www.clontech.com/NL/Products/Viral_Transduction/Selection_Guides/Lentiviral_Workflow?sitex=10140:22372:US

Hope this will also help other researchers having similar problems.

Thanks and regards

Rajendra

Dear all,

Thank you for valuable inputs.

I was able to transduce my target cells and had about 50 % cells expressing GFP. However, it is not ideal for my work as the expression of GFP was dependent on time. atleast it took 6 days. Therefore I had successfully generated Tet-On inducible plasmid and was able to overcome the odds of transduction.

Thank you again.

Good luck.