7 replies to this topic

[medo]

Hello all,

I am transfecting 293T cells with packaging lentiviral plasmids to generate a lentiviral particles carrying GFP and another gene. When I infect 293T cells with the viral particles to calculate the viral titer I get a very high efficient GFP signal on FACS. But the problem is, when I infect mice spleen cells specially MACS separated B-cells as my 1st interest nothing really happens !. Anybody ever used lentivirus for transduction of cells ? Do you have any suggestions to imprvoe the infection of the cells?

Cheers.  

Edited by medo, 20 May 2009 - 07:42 PM.

[Clare]

Hi there

I haven't used lentiviral infections but have done plenty of retroviral infections of B cells. What B cells are you trying to infect? Are you stimulating them prior to infection? What method of infection are you using?

Clare

medo, on May 21 2009, 04:31 AM, said:

Hello all,

I am transfecting 293T cells with packaging lentiviral plasmids to generate a lentiviral particles carrying GFP and another gene. When I infect 293T cells with the viral particles to calculate the viral titer I get a very high efficient GFP signal on FACS. But the problem is, when I infect mice spleen cells specially MACS separated B-cells as my 1st interest nothing really happens !. Anybody ever used lentivirus for transduction of cells ? Do you have any suggestions to imprvoe the infection of the cells?

Cheers.  

[medo]

Hi Clare,

Thanks. I separate B-cells from mice spleen using MACS CD19+ microbeads, plate them on 12-well plates ( 1 million cell/well ) overnight and then infect them with concentrated viral supernatent in the presence of 8 micrograms/ml Polybrene and analyze for GFP expression after 72 hours of incubation. This method works very well on 293T cells but not on B-cells and spleen cells in general!. I dont stimulate the cells, do you think its important to do so?

Best regards,

Medo

Edited by medo, 21 May 2009 - 03:28 AM.

[Clare]

Hi Medo,

Have you tried doing a spin infection? I found this was better than just culturing in polybrene/virus. It gave me a much better % of GFP+ cells.

In some of my experiments I did stimulate them as I was interested in what effect overexpressing a particular gene would have on antibody secretion. In these experiments I used resting B cells.

What exactly do you want to study in your B cells?

Clare

'B cells are brilliant, T cells are terrible!'

medo, on May 21 2009, 12:25 PM, said:

Hi Clare,

Thanks. I separate B-cells from mice spleen using MACS CD19+ microbeads, plate them on 12-well plates ( 1 million cell/well ) overnight and then infect them with concentrated viral supernatent in the presence of 8 micrograms/ml Polybrene and analyze for GFP expression after 72 hours of incubation. This method works very well on 293T cells but not on B-cells and spleen cells in general!. I dont stimulate the cells, do you think its important to do so?

Best regards,

Medo

[gfischer]

The first thing I would do is check the literature for people who have done transduction of B-cells.  I suspect that the promoter/envelope/packaging construct you're using just isn't working well in your cells.  See if others have already done it and see if their virus differs from what you're using.  If no one has done this type of transduction, it would be worth your while to try a variety of promoters and envelopes to see what combination works best.

Good luck

[medo]

[quote name='Clare' date='May 21 2009, 01:03 PM' post='24855']
Hi Medo,

Have you tried doing a spin infection? I found this was better than just culturing in polybrene/virus. It gave me a much better % of GFP+ cells.

In some of my experiments I did stimulate them as I was interested in what effect overexpressing a particular gene would have on antibody secretion. In these experiments I used resting B cells.

What exactly do you want to study in your B cells?

Clare

'B cells are brilliant, T cells are terrible!'



Thanks Claire. I did a spin infection once but on sterile FACS tube. Incubating the tube overnight and then transfer the cell suspension to 12-well plate but this didnt work. I will try the spin infectin on the plate itself as you suggested and hopefully this will solve the problem.

I am trying to do an in-vitro knockout on immune cells using lentivirus. My gene of interest already flanked with two loxP sites in all mice cells so when I add the viral particle carying Cre recombinase this will result in deletion of my target gene. After achieving this knockout system I am going to test the effects of deletion of this gene on the immune cells starting from B-cells cuz they are brilliant as you said lol.

Regards

Edited by medo, 21 May 2009 - 09:13 AM.

[medo]

gfischer, on May 21 2009, 03:28 PM, said:

The first thing I would do is check the literature for people who have done transduction of B-cells.  I suspect that the promoter/envelope/packaging construct you're using just isn't working well in your cells.  See if others have already done it and see if their virus differs from what you're using.  If no one has done this type of transduction, it would be worth your while to try a variety of promoters and envelopes to see what combination works best.

Good luck

Thanks, I searched the net and found only one paper which they are doing the same thing as I do ( the same lentiviral vector and packaging plasmids ) but the only difference is that they are doing spin infection of the plate at 700 g for 1 hour. I hope this will work otherwise as you suggested we will try to change the packaging plasmids.

Regards.

[Clare]

Hi again

Did you want my spin infection protocol? For the B cell infections, I spun the cells with the virus in 15ml round bottom tubes. I found this was better than culturing on 6-well plates (in which the virus has been spun down onto).
Clare