Hi!
I am trying to select the lowest amount of blasticidin needed for selection in murine primary neurons. I have tried concentrations from 1-8 ug/ml and had no difference in the fitness between the cells. The cells did appear dead, the axons were disintegrating but the cells didnt actually detach from the wells.
I am not sure what to make out of this, could I use blasticidin for selection and passage the cells & count on that the dead cells wont attach but that the live ones will?
Any input would be appreciated.
Best,
Linn
Hi!
Hard to help give ideas without knowing the exact protocols. So, I don't know if this will be helpful, but:
Did you transfect primary cells post-isolation? Or, do you have a knock-in/KO mouse line?
Assuming the former: How tricky is it to isolate and transfect those primary cells for you (costs and time)? IF you can manage, have you tried transfecting at a lower vector titres, then selecting at smaller intervals of [blasticidin]? I don't know if your cells are sensitive to agitation, but during transfection, you can put the sample dish/whatever on an agitator or make sure the solution is well mixed.
If nothing works out (as I don't know exactly what you are working with), my friendly suggestion is this: You might want a "clean" non-transgenic mouse, try to transfect post-isolation of primary neurons (sounds tricky already), because it might be easier and less expensive than producing new transgenic mice. And, you can titre your vector at differing concentrations to at least get whatever protein expressed...However, this is only a first step and its hard to draw conclusions on such a experimental design. So, your holy grail would be: transgenic mice homozygous for whatever gene ki/ko of interest is the best for meaningful data (as you may already know and appreciate).
Primary neurons research is tricky! Good luck!
I would also propose to give some more information regarding the whole experimental setting.
Hi Prasanth and Andreas,
I will provide more info on the experimental setting. So I am working on wild type mice and the neurons are first isolated and plated. Afterwards I am aiming to transfect them with 1) pX330 plasmids expressing cas9 and the gRNA for my specific target and 2) a selection vector harboring the sequence that the gRNA should recognize and cut. It also contains a frameshifted non-functional blasticidin resistance gene. When the cas9 cuts the sequence on the plasmid a frameshift will repair the blasticidin gene.
Before starting the experiment I want to find out which is the lowest concentration of blasticidin needed to kill cells without blasticidin resistancy. I tried concentrations ranging from 1-8 ug/ml and they all had their axons degrading and looked dead or dying atleast but did not detach from the well.
In this case, can I trust that this kind of selection would work? That when replating them the transfected neuron with the resistance gene would again attach in the new well but that the dead ones would float? Or should I just go higher in blasticidin resistance risking toxicity to my transfected cells?
Best,
Linn
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