Hello everybody,
I Need two generate 2 New cell lines, using iPSCs to express a calcium indicator (GECI) in one of them and channelrodopsin (chr2) in the other one.
I am not anche expert and i am trying to understand if is better through the creation of a lentivirus, or using Crispr/cas9 technique.
What's the most effective strategy and someone can explain me the pro and cons of each of them?
Thank you
By Crispr, do you mean dCas9-VP64 for to activate it? If yes, just overexpress your genes directly with the lentivirus. Using dCas9-vp64 is mostly diffucult to optimize and unlikely to work great w/o off-targets.
If you just need to integrate your calcium sensor and your channelrhodopsin but have no need to insert it in a site-specific manner, then a lentivirus vector will do well - you might even be able to introduce them in the same vector as long as the combined sequence isn't too long
This is not an either or question. Lentivirus is a way to put your genetic construct into the cell. CRISPR/CAS9 is part of your genetic construct. For example, you could use lentivirus to put CRISPR/CAS9 into the cell. (subject to size limitations)
As others have suggested, try to specify in detail what you want to do. Then, we can think about which tools might help you do it.
First of all thank you all for your answers.
In detail I have to create IPSCs with a stable expression of Channelrodopsin and GECI downstraim a CAMKII promoter. I don't need to insert it in a site-specific manner
My first idea was to design the plasmid to be delivered via lentivirus for both of cases. I was concern about the efficiency of the trasfection and of the final result. So that I was looking at the idea but I have read about the limitation of the insert dimension and about the difficulties in isolating single IPSC to isolate the clones for the selection and the final expansion. For this reason I was asking for an expert suggestion to understand what could be the best approach. Again, I am not an expert in this and my lab is not focused on this topic.
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