7 replies to this topic

[gold brook]

I was trying to make some retrovirus for labeling neurons in cortex. But I couldn't get it work at the first step. I can't get the virus.
Here is what I did:

Carrier vector: pQCXIX (inserts has RFP);
Cell line: GP2-293 or 293T
Transfection methods: Calcium phosphate or Fugene 6

One day after transfection (pQCXIX and pVsVg for GP2-293; additional pcmv-GP added for 293T), I can see very good red signals in cells. 2-3 days after transfection, I transfered 1 ml supernatant into NIH3T3, there was no virus at all, no matter how long I waited. I also tried ultracentrifuge the supernatant and still nothing.

What could be wrong here? It's quite frustrating. Please help! Thanks!

[bob1]

Did you look at your cells?  Were they lysing from the virus exiting the cell?  If not, you probably didn't have much virus in the medium/supernatant, try leaving the 293 for longer.

[stardust]

Hi,

is it enough to transfect the retroviral plasmid and the VSV-G? Where does the gag and pol come from to produce virus? I don't think 293T express these viral genes...Oh sorry, I did not read your post properly, you also transfected gag and pol...could you maybe attach a picture of yoiur cells after transfection showing the RFP? What do you mean by saying you transferred 1 ml to nih3t3 and there was no virus? How do you determine the viral titer?

Stardust

Edited by stardust, 02 October 2009 - 05:02 AM.

[gold brook]

When calcium phosphate used for transfection, most of cells would got detached after 2-3 days. With Fugene 6, most of cells are still fine on the plate. The longest time point I waited was 4 days after transfection. Right now, I think the system is not making any virus at all. The time window will just affect virus titer, not all or none, right?

bob1, on Oct 1 2009, 04:18 PM, said:

Did you look at your cells?  Were they lysing from the virus exiting the cell?  If not, you probably didn't have much virus in the medium/supernatant, try leaving the 293 for longer.

[gold brook]

Hi, Stardust,

2-3 days after transfection, I always saw at least 60% of cells were expressing RFP. (But I didn't take any image from them.) Then I collected the medium and filtered it with 0.45um filter. At the end, I added 1 ml filtered medium to NIH3T3 splitted a day before. After several days, there was no cells showed RFP expression.
Right now, I am trying to get virus with RFP only (in MCSII) from this system. If it works, then that means my insertion (in MCSI) caused this no-virus situation, although I don't like this explanation.  

stardust, on Oct 2 2009, 05:57 AM, said:

Hi,

is it enough to transfect the retroviral plasmid and the VSV-G? Where does the gag and pol come from to produce virus? I don't think 293T express these viral genes...Oh sorry, I did not read your post properly, you also transfected gag and pol...could you maybe attach a picture of yoiur cells after transfection showing the RFP? What do you mean by saying you transferred 1 ml to nih3t3 and there was no virus? How do you determine the viral titer?

Stardust

[stardust]

For titer determination, we usually include a selectable marker in the retro- or lentivirus. Then we select for 2 weeks and count the colonies.

However, if you can not see RFP in the infected cells, you might be right with your guess that you have not enough virus. Or perhaps the virus is not very good for infecting your cell line of interest although you are using VSV-G envelope. It might also be possible that not enough copies of the virus integrate to see RFP. Maybe you could isolate RNA and do an RT-PCR for RFP to see if any cells got transduced?

But i guess it doesn't help if you want to infect your cells if there is not enough virus...and how big is your construct (between the LTRs)?

Personally, I always use calcium phosphate method because for me Fugene doesn't work (but I mainly produce lentiviruses) and I usually harvest the virus after 48 h.

Is your gene known to be toxic or something?

In which format are you performing your transfection? 10 cm dish? How much medium?

And the transduction with the virus? 6 well plate? How much medium?

Stardust

Ah and which promoters are in front of your gene of interest and RFP?

Edited by stardust, 02 October 2009 - 08:07 AM.

[gold brook]

stardust, on Oct 2 2009, 09:06 AM, said:

For titer determination, we usually include a selectable marker in the retro- or lentivirus. Then we select for 2 weeks and count the colonies.

However, if you can not see RFP in the infected cells, you might be right with your guess that you have not enough virus. Or perhaps the virus is not very good for infecting your cell line of interest although you are using VSV-G envelope. It might also be possible that not enough copies of the virus integrate to see RFP. Maybe you could isolate RNA and do an RT-PCR for RFP to see if any cells got transduced?

But i guess it doesn't help if you want to infect your cells if there is not enough virus...and how big is your construct (between the LTRs)?

Personally, I always use calcium phosphate method because for me Fugene doesn't work (but I mainly produce lentiviruses) and I usually harvest the virus after 48 h.

Is your gene known to be toxic or something?

In which format are you performing your transfection? 10 cm dish? How much medium?

And the transduction with the virus? 6 well plate? How much medium?

Stardust

Ah and which promoters are in front of your gene of interest and RFP?

Hi, Stardust,

I tend to believe that something is not working in my carrier vector. The length between LTRs in around 6.3kb, which should not be an issue. The insertions in MCSI is definitely not toxic to cells. But I am worrying about some specific secondary structure formed which might interfere with the packaging of virus. I usually perform transfection with 10cm dish. Before transfection, I put 9 ml fresh pre-warmed medium to cells, and then later add 1 more ml medium containing calcium phosphate-DNA particles or Fugene6 with plasmids. I tried 24-well, 12-well (1ml), 6cm dish (5ml) and 10cm dish (6-10ml) for transduction. None of them showed anything. It's very annoying. The vector I am using is pQCXIX. So RFP is under IRES and gene of interest is under CMV or CAG. CAG should be strong enough to drive my gene expression if everything worked out and also help RFP expression.

Gold brook

Edited by gold brook, 02 October 2009 - 09:10 AM.

[PauGF]

Hi there,

You should consider if your target cells are actively dividing to allow retroviral transduction. I tried to transduce with the same supernatant B16 cells and activated murine T cells... only the latter ones expressed my gene of interest. Be careful with FBS, cause interfers with polybrene, and with the differences in the medium of target cells and packaging cells.

Regards!