2
$\begingroup$

I have been having difficulties with low transduction efficiencies of my retrovirus production. I expand my plasmid of interest (on MiG-GFP plasmid) in DH5α E Coli for ~24 hours, purify with Qiagen midi prep kits, then put on ~90% confluent 293FT cells with either retronectin or polybrene for 24 hours. I then change media and harvest supernatant at 48 and 72 hours of incubation with the 293FT cells. I then expose 3T3 cells to varying volumes of the virus containing 293FT media and am able to transduce about 10% of the 3T3 cells, which is really low. That being said, here and there I am able to get very high transduction efficiencies on the 3T3 cells, but for unknown reasons. I have tried concentrating the virus but transduction efficiencies don't increase all that much. And I have tried growing up the plasmid in Stbl3 cells, which if anything gives me worse virus.

Anything here jump out as being responsible for low transduction efficiencies. Or is there another way somebody does this that works really well?

$\endgroup$
  • $\begingroup$ Have you tried using a transduction agent? I personally suggest effectene if you decide to try your luck there. What titre are you using? How much did you change the titre when you concentrated it? $\endgroup$ – CDB Apr 13 '15 at 17:25
  • $\begingroup$ I also use polybrene as a transduction agent and have used 5-FU to get my cells cycling prior to transduction. But I have not tested effectene, I appreciate the suggestion. And when I concentrated the virus the titre tested on 3T3 when up by about 5-10% with what I would have expected to be a 10X concentration. $\endgroup$ – The Nightman Apr 13 '15 at 17:54
  • $\begingroup$ Hmm, that titre should definitely have increased the efficiency by quite a bit. I'm not sure what could be restricting that. Well if you want to keep the retroviral vector (of course effectene is only good for free DNA) there are several reliable brands of transduction agents you could try. $\endgroup$ – CDB Apr 13 '15 at 18:36
2
$\begingroup$

In my experience, transduction efficiencies skyrocketed when I applied a spin-infection procedure. A 30 min low speed centrifuge with the virus changed a lot. Here is the TRC protocol for this: http://www.broadinstitute.org/rnai/public/dir/download?dirpath=protocols/production&filename=TRC%20viral%20infection%20200909.pdf

$\endgroup$
  • $\begingroup$ I have tried this. I have even spinfected for 2hr followed by a 3hr 37C incubation and it does make a slight difference, but still doesn't solve the problem. $\endgroup$ – The Nightman Apr 13 '15 at 19:17
  • $\begingroup$ In this case, using ideal (nontoxic) dose of polybrene and spinfection, I'd look for an optimal stage on the growth curve of your 3T3s. It's been a while since I've worked with that line. But in other cases of transfection, establishing the curve on uniformly spread cells (with polydL coating or so) helped a lot. (I am aware of transfection&transduction differences, yet I find that fast growing helps with the latter as well. $\endgroup$ – WesternBlöd Apr 16 '15 at 4:16
  • $\begingroup$ Also, try titering your virus in HEKs or HeLas, just to make sure that your production is not the issue here. Protocol here: ow.ly/LFQHe There are qRT-PCR titering methods as well saving effort; such as this one:ow.ly/LFQK2 $\endgroup$ – WesternBlöd Apr 16 '15 at 4:27

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.