Can somebody please explain to me the difference between these two?
I'm aiming to inhibit neuronal firing with Clozapine N Oxide (CNO) after the successful transfection and expression of a G(i) coupled DREADD. I'm growing primary hippocampal cultures from WT rat pups and would be intending on introducing the plasmid vector (with gene of interest) to the neurons using Lipofectamine 2000. In what respect would Cre dependent versus Cre independent DREADDs have on the levels of expression?
To give an example, I'm currently trying to compare Roth labs:
AAV-hSyn-DIO-hM4D(Gq)-mCherry (Cre dependent)
vs
AAV-hSyn-HA-hM4D(Gi)-IRES-mCitrine (non-Cre dependant)
http://blog.addgene.org/plasmids-101-cre-lox
Here's a start towards trying to understand why you'd want to consider Cre vs. non-Cre dependent constructs.
Hi Christopher,
in the situation you described a NON-Cre dependent version is the easiest approach. Basically the Cre dependent versions will only be expressed when Cre recombinase is present in the infected cells; since you mentioned that you are using WT animals a Cre dependent DREADD will just not work in your system (unless you co-express Cre).
One option you could consider instead of transfection is to buy the packaged virus or package it yourself as you will get a higher number of expressing cells. Even though this would be more expensive than the transfection could be worth depending on the specific study you want to do.
Hope this helps,
Ezequiel
Thank you both for your answers, I'll read over the addgene info prior to my meeting with the supervisors.
Ezequiel, am I right in saying then, were I to use a Non-Cre dependent vector and target neurons specifically within a mixed culture (glia included), I should be looking at a transgene that is promoted by a neuronal specific protein such as Human
Synapsin 1?
Hi Christopher,
as you mentioned the specificity of the expression of the DREADD in a Non-Cre dependent virus will depend in the promoter that drives the expression but also by the tropism of the virus (if you use infection instead of transfection); different serotypes will be able to infect different cells with a varying degree of efficiency. AAVs serotypes 5, 8 and 9 are the most common in neurons and are very good at it however I do not know if they would be able to infect glial cells. However since the promoter is neuronal specific you should be fine. Another point to consider is that the DREADD will be expressed both in pyramidal cells as well as GABA interneurons and this could have an effect in the outcome of your experiment depending on the methods you use, for example western blot, where you analyze all the cells vs, electrophysiology where you just select one in the coverslip.
Hope this helps,
Ezequiel
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