4 replies to this topic

[loucash]

Hello everybody

I am currently trying to establish lentivirus production in HEK293T cell line. I have two packaging systems I could use:

• pLP1 / pLP2 / pVSV/G   (from Invitrogen i think, we somehow have the plasmids separately)
  
• pCMVR8.92 (encodes gag/pol) / pRSV-rev (rev) / pMD.G (VSV env)

pCMVR8.92 is a gag-pol vector similar to pCMVR8.91 with a deletion in the Rev coding sequence afaik.

I have three different transfer vectors i would like to use:

• pLVX-dd-ZsGreen1 (Reporter System from Clontech)
  
• pLKO.1 (commonly used shRNA vector)
  
• pMF359-IRES-EGFP (constructed lentiviral vector based on pNL-EGFPU3, see this publication: http://nar.oxfordjou...e113#GNF112TB1)

I seeded 7*10^5 HEK293T cells on a 3.5cm dish (6-well plate) and on the next day i did transfection with MetafectenePRO (Biontex) with following DNA amounts:

1ug of all packaging plasmids and env-plasmid, 2ug of transfer vector (I only tried pLVX and pMF so far)

Transfection worked since i had green cells the next day (when I also changed the media). Efficiency was around 60% (of course i can only say this for the transfer vector).

Unfortunately the harvested supernatant could not infect HEK293T or human melanoma cells (I added 200 ul cleared but unconcentrated supernatant to 80% confluent cells in a 6 well plate or 1.2 ml sup to 100mm plate)

Does anyone see an obvious error? Why do most protocols use Calcium-phosphate method for the transfection? I have a protocol from my lab which uses FuGene, but I think this is basically the same as MetafectenePRO.

What about the plasmid ratios? What i have seen online (e.g. lentiweb.com) people apply much transfer vector, less packaging plasmids, and even less envelope plasmid. Why? wouldn't it make more sense to make sure that every cell that takes up the transfer vector really contains the packaging and env plasmids? (e.g. make a ratio of 1:5:5:5 of transfer-vector:packaging-plasmids)

Any further suggestions?

Thanks for any answer.
Lucas

Edited by loucash, 02 June 2010 - 01:11 AM.

[laurequillo]

I do the transfection with Rotifect, but whatever reagent you have is ok. Here the ratios I use (6cm dishes):
- 3.5 ug shRNA (PLKO.1)
- 2.5 ug psPAX2
- 1 ug VSV-G


It looks like you have problem producing the virus. Anyway I will try first to go up with the volume of virus you are using to infect the 293T cells (0,100, 300, 800, 2000 ul) and I´d use 50% confluent cells.

Edited by laurequillo, 02 June 2010 - 05:49 AM.

[loucash]

Thanks for your answer.

I tried infection with 1 ml filtered supernatant last week and again got no green cells. I am thinking there has to be something basically wrong. It seems that no virus is produced at all.

My expectation would be that most of the things (like plasmid ratios, DNA:Metafectene ratio, time points of medium exchange) one can adjust do only influence the final titer but do not result in no green cells after infection at all..

I am a little bit clueless what else I could try

[laurequillo]

So, you transfect the cells, and 48h later you collect the supernatant, you add polybrene (or not) and then you freeze it in alicouts, right? and you check the infection on 293t cells. Well, if you dont see any green cell I´d say you are not getting viruses, but I dont know what´s wrong...Are you sure you are using the proper plasmids??

[zodiac1505]

We use lentiviral transgenesis in our lab and sometimes it works great and sometimes it doen't work at all. We don't use the exact system but I thought I could outline the protocol for you so that you could have an idea. We use our own plasmids and usually make virus from 20 cm plates of 293 cells. We use 20 microgram plasmid with 3 microgm VsVg, 3 microgram RSV, 4 microgm pMDL and 5 microgm pAdv. We collect the 48 hour supernantant, spin it down, filter and ultracentrifuge it and leave it overnight in 100 microlitre PBS. Aliquot it and use 1 microlitre and dilutions to titrate the virus. The titration is done on 293 cells in a 6 well plate.

The transfections always look great on the 20 cm plates but sometimes we don't have any green cells on titrations. We have tried several things to sort this out. I can suggest a few though it might not be the case with you. (1) Maybe you need to thaw new 293 cells and use them (2)Try making new media (3) Maybe one of the plasmids is defective. Sometimes we had to borrow plasmid components from others and make new maxis and then we started getting virus (4) Change the transfection reagent. We use several ones but Fugene HD and Genejammer works best for us. Hope this helps.