Lentivirus expression system - get no or low titer of virons (Jun/29/2009 )

Hi everyone,

I have some questions about producing virus via Lentivirus system.
I cloned big insert (~3.4kb, with EGFP at 3') into lenti-based vector and co-transfected with package plasmids into 293FT cells. After 48~72hrs, I collected supernatant and titered by infecting 293FT. However, no GFP could be seen. I am sure that 293FT transfection is successful since 293FT with many GFP fluorescence. So, I guess the problem is viron could not be produced. Any suggestion for packaging the large protein?

mydove on Jun 29 2009, 06:58 PM said:

3.4kb does not sound unreasonably large to me.. how large is your vector in total?
are you sure your packaging plasmids are working (ie can you make high titer virus of other vectors?).
can you package the empty GFP vector with reasonable titer?

also check the purity of your plasmid of interest prep to make sure this isn't your problem (bad preps can reduce virus titer quite a lot in my experience).

which lenti vector is your insert in?

Many thanks for your comments.
I utilized pLKO-AS2 as lenti-based vector. My target gene is N-cadherin with eGFP at 3' end.
http://rnai.genmed.sinica.edu.tw/file/vect...LKO_AS2_Map.pdf
So the construct I made is around 10kb.
Yes. I could produce eGFP virons by the same procedure. Unfortunately, it doesn't work when i produce the larger one.
I will check the plasmid (Lenti-vector and Package plasmids) again.

mydove on Jul 5 2009, 07:36 PM said:

I am using a 11kB lenti-vector (pLenti6) regularly and that usually works fine. Our empty pWPI-ires-EGFP is 12kB or so and also packages without problems.
pLKO I only tested something close to 9kB which also worked - therefore I have my doubts that size is the problem.

Thanks for your help.

Actually, I also cloned another gene (~1.8kb) into pLKO-AS2 to get ~8.6kb plasmid. This construct could be packaged successfully. I don't know why it doesn't work when the size is increased to 3.4kb (or even 2.7kb).
I re-checked lenti- and packaging plasmids. There is no problem with these plasmids.

It seems that I have to buy lenti-vector from "Invitrogen"? So expensive

Sometimes it's not the size of the insert, it's the gene of your interest. For example a genes expressed in the packaging cell line somehow down regulates the level of lentiviral trascription, therefore less lentivirus will be made.

If you want to buy pLenti from Invitrogen, make sure you get pLenti6.3 which contains cPPT and WPRE that will increase your gene expression ~10-fold. However, there is not much advantage of using pLenti6.3 since many lenti vectors either in other labs or commercially available also encoded cPPT and WPRE. In you case, one of the problems is "293FT". Try 293T, your virus titer might be 10-fold or more up.

Regarding the protocol, send me an email if you need one.

Good Luck.

Hi Mydove,
Just to let you know, Invitrogen is not your only option for lentiviral vectors! ABM offers lentiviral vectors, and can even custom make them for you at a much lower price than invitrogen. You should check them out, their web page is www.abmgood.com

Jennlou2

Okay.
Thanks for your information.

Correct me if am wrong! isnt pLKO vectors with U2 promoters meant for small shRNA molecules?

Dear Molonco
You are right. pLKO was first designed for cloning shRNA. pLKO.AS2 is an modification of pLKO. People removed U6 promoter, WPRE and cPPT sequences and added CMV promoter instead.