I have already optimized protocol fro generation (I work every day with Lentivirus in my lab) but I have never infected hESC and I don't find any thing in literature until now.
you could use polybrene when you transduce iPSCs with lentivirus. It helps in better transduction. Other than that, you just need to add the virus to the cells and incubate for 24hrs depending on your virus. Sometimes a double infection increases the efficiency. It is better to use fresh polybrene everytime you do the transduction.
for polybrene I use 6ug/ml. As for the MOI 10 would be optimum.
Hi , did you succeed? I tried to do it and nothing working for me. I have a good infection of mefs but not of iPSC.
Hi, I tried to transduce without polybrene (it is toxic for IPSc) at MOI 10 and it is good. I worked in feeder free condition. In this way you don't have the problem of Mef but the cells are more susceptible at death. You have to find a good concentration of cells to prevent the apoptosis (use Rock inhibitor obviously) and to maintain the undifferentiated state.
TNX
Will try because it did not work for me before
last question, you know the name of your promoter in lentivirus?
In general I use a Tet On system so I perform a double infection with LV:pPgk1-rtTA2S-M2 and LV:TREt (gene of interest)
Just a few notes for both of you:
Polybrene shouldn't be toxic to hES cells at all. Check your concentrations. It is only charging the surface to enable lentivirus to bind better.
Smaller colonies are easier to transduce.
Promoter is important. I you have feeder in the culture, they will often be much brighter and hence it looks like the ES cells are not transduced. However, they can be transduced albeit a much lower level of expression. That is why MOI 10, as Clara mentioned, is often used to hit more cells and get more copies per cell to increase expression.
Maybe a few useful references: 1. Liew C-G, Draper JS, Walsh J, Moore H, Andrews PW. Transient and stable transgene expression in human embryonic stem cells. Stem Cells. 2007;25(6):1521–1528. doi:10.1634/stemcells.2006-0634.
1. Zhou BY, Ye Z, Chen G, Gao ZP, Zhang YA, Cheng L. Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. Stem Cells. 2007;25(3):779–789.
Thank you Christian. I already received a good transfection after I change to another promoter!
Can you explain to me why it can be look brighter if I have a feeder?
Thank you
Usually fibroblasts are much bigger cells and will get hit by many more virus particles during infection. So you will have more copies in the fibroblasts and your promoter expression is often much stronger in the fibroblasts too. I hope that was an answer to your question. If not please clarify.
My experience is polybrene is non-toxic to the hiPSC. The problem I have is the transfection. I transfected the human iPSC with lentivirus (FIV3.2CMVCre-eGFP VSVG), but only the differentiated cells are transfected with 100% efficiency. None of the stem cells had been transfected. Should I try another virus?
The stock viruses in our university core are almost CMV promoters (https://vector-core.medicine.uiowa.edu/collections). Do you know any commercial virus that is known to work well in human iPSC?
Appreciate your input!