Hi,
I want to infect my CD34+ derived human iPSC with shRNA library, and to determine at which MOI values to test, I did some search but could not find much information. Has anyone transduced (human) iPSC so far? Are they easy to transduce or would one have to infect at high MOI?
Dear Ceren,
many parameters would influence the infection/transduction efficiency of virus (sequence design, viral particle used, permissivity of the cells...), and it is thus difficult to answer correctly to your question,
However, I would propose you to try two methods that allows to lower the MOI and enhance the viral particles efficiency. In this way, infection will be facilitated (even for non-permissive cells) and less material can be used for the same efficiency. In general these methods allows using MOI from 0.01 to maximum 5 ot 10.
- The first one would be a direct substitute to Polybrene: LentiBlast. It neutralizes electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Moreover, due to a favorable “membrane permeable effect” limiting the transmembrane potential changes it is non-toxic and totally compatible with cell viability.
- The second one would be the Magnetofection technology *** with ViroMag and ViroMag RL. These two magnetic particles formulations bound to viral particles and allow a dramatic increase in infection and transduction.
I'm pretty sure these reagents will give you reliable efficiency, you can contact me directly at [email protected] or via Researchgate if you need more informations or if you want to try either LentiBlast or Magnetofection.
Good luck for your experiments,
Cedric
*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).
Hello
find attachment & links here
Good Luck
http://www.cell.com/cms/attachment/598577/4701300/mmc1.pdf
http://www.biomedcentral.com/1471-2121/13/9
http://www.nature.com/mt/journal/v20/n1/full/mt2011234a.html
http://search.proquest.com/openview/5ef66e36f61f8bf910338d9b452bf4bb/1?pq-origsite=gscholar
Thanks for both answers but my question was not about reprogramming as I already have reprogrammed, iPS cells. I would like to transduce these iPSC with shRNA constructs, and wanted to learn whether iPSC are easy to infect or one would need to use virus at high concentrations/MOI with a transduction reagents, etc.
Hi Ceren: Treat the iPSCs like you would treat any other target cells. Only difference would be your pluripotent media and this should not interfere with your lenti-transduction. I have transduced iPSCs with lentivectors and achieved quite good (> 80%) transduction efficiency without any problem. It is advisable not to use polybrene to enhance transduction as this may interfere with the components of your pluripotent medium. Cheers.
Good Morning Ceren Günes
which MOI was needed for your iPSCs?
All the best,
Julie