Hi Christina,

In general primary murine lymphocytes are a pain to transduce.

In several publications it is common to refer to 30% positive as pretty good so I would start with low expectations and work up from there.

Few questions: How did you check your transduction efficiency? How did you run your spinoculation protocol? What protocol do you use for transfections and on which cells?

I heard that LentiBoost is very good (haven't tried it yet but soon) and I think it would be a good direction.

I too never heard about working with un-activated splenocytes, can you keep them in culture long enough for you experiment?

Good luck,

Or

Hi Christiana,

I have previously commented on this issue; but the short answer is that murine T-cells don't process lentiviruses.

Article Genetic Engineering of Murine CD8(+) and CD4(+) T Cells for ...

You also have to consider how you will keep mature T cells alive in culture without stimulation.

A big thank you to everyone for taking the time to reply to my question!

Or Carmel

How did you check your transduction efficiency?

The lentivirus vectors encode for different fluorescent proteins which I detect by flow cytometry.

How did you run your spinoculation protocol?

In short:

• isolate fresh splenocytes
• resuspend cells in medium containing "Viral Entry" (ABM)
• add lentiviral particles, mix by tapping, let sit at room temp. for 10 minutes
• spin at 2000x g at 30°C for 1h (in 1.5 mL tubes)
• resuspend and transfer into 96 well plate and incubate
• repeat the spinoculation 24h later but in the plate (Take out 1/2 of the medium and replace with "Viral Entry" and Lentivirus containing medium)

What protocol do you use for transfections and on which cells?

• we purchased manufactured lentiviral particles so we didn't need to do any transfections

I too never heard about working with un-activated splenocytes, can you keep them in culture long enough for you experiment?

• I hope so, I am planing to add IL-2 to the culture

Lillian Onwuha-Ekpete

The problem that I am having here is that I am trying to transduce resting cells, which to my understanding won't work well with gammaretrovirus. That's why we chose to use lentivirus. Further, the lentivirus is ecotropic for mouse cells, which might facilitate at least the entry. If we can't make the lentivirus transduction work though, we might have to switch to a retroviral vector system.

Wooseok Seo

I have whole splenocytes, so in addition to adding IL-2 the next time, I am hoping the additional cells in the mix will provide some survival signals.

Again, thank you all for your input. I am happy to hear any further comments and thoughts on the matter.

Christina

Hi Christina,

Your measurement protocol is similar to mine and I think it should work fine.

For your spinoculation:

Make sure you calibrate the amount of splenocyets, in my hands it have a big impact (I work with 6 and 24 wells so my numbers will differ from yours).

I tried only once to infect cells in a 1.5ml tube (spinning the plate is much more common, the surface area for interaction with the virus is larger). In my experience you don't need a long centrifugation with the cells, 10 minutes should be enough. Did you see good amount of live cells after this centrifugation?

Another thing that I tried is to keep the cells after centrifugation with the viral particles for 24h without re-suspension.

Did you tried to infect "easier" cells (293T) with this virus? just to make sure if there is an issue there.

Splenocyets do help with CD80\86 but with no CD3 engagement I don't know for how long they will survive ex-vivo. I also heard that DC's and supportive splenocyets mostly die in 24h.

I'm too trying to improve and solve this problem, and will update if I get better results.

I hope this is helpful and wish you luck,

Or

Thank you, Or Carmel . I will certainly take your input into consideration. I will perform a "dry run" without virus next week. Just comparing different protocols and see how harsh the different treatments will be on the cells. The centrifugation times range from 30 to 90 min, I'll also include 10 min. I had the virus left on the cells before and will do so again.

In the end, our plan is to leave the cells in culture as short as possible, probably less that 24h, what ever shortest period of time might be sufficient for transduction.

I actually saw GFP+ cells, however none of them were CD4+. So I think the virus is fine, it just didn't enter the T cells well.

Thanks again, I'll keep you posted on what I find.

Christina