Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Lentivirus expression system


  • Please log in to reply
10 replies to this topic

#1 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 29 June 2009 - 05:58 PM

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?

#2 warsel

warsel

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
0
Neutral

Posted 01 July 2009 - 03:21 AM

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?


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?

#3 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 05 July 2009 - 07:36 PM

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.s...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.

#4 warsel

warsel

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 77 posts
0
Neutral

Posted 06 July 2009 - 06:56 AM

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.s...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.


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.

#5 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 06 July 2009 - 09:44 AM

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 :)

Edited by mydove, 06 July 2009 - 09:45 AM.


#6 Functional Screens

Functional Screens

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 118 posts
5
Neutral

Posted 10 July 2009 - 10:36 AM

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.

#7 jennlou2

jennlou2

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 16 July 2009 - 02:42 PM

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

#8 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 17 July 2009 - 10:18 PM

Jennlou2

Okay.
Thanks for your information.

#9 calvin*

calvin*

    Calvin*

  • Active Members
  • Pip
  • 19 posts
4
Neutral

Posted 18 July 2009 - 05:37 AM

Correct me if am wrong! isnt pLKO vectors with U2 promoters meant for small shRNA molecules?
"In my opinion, we don't devote nearly enough scientific research to finding a cure for jerks"-Calvin

#10 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 19 July 2009 - 03:37 AM

;) 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.

#11 mydove

mydove

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 29 July 2009 - 09:39 PM

Dear all,

Unfortunatelly, it doesn't work when I used 293T as viral production cell line.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.