Iontophoretic AAV injections

Science
1

Hey guys,

I’m a postdoc who’s been using the Allen Institute’s protocol for iontophoretic injections of AAVs for some years now ( Stereotaxic Injection by Iontophoresis V.8 protocol abstract ). Great protocol, works very well for localizing injections and preventing expression in the pipette track etc.

My issue is that Stoelting is no longer manufacturing the Midgard Precision Current Source, nor are they servicing existing instances. First of all, I’m curious what people at the Allen, or elsewhere, are using nowadays for this method.

Second, the reason I’m looking into a new current source is because I’ve been having some crazy issues with my Stoelting box over the past year. In short, injections seem to be failing - no expression 4 weeks later - without any indication of a clog in my pipette or other interruption in the circuit (that is, there’s no voltage overload indicated on the screen, and voltage during applied current appears pretty consistent). When I see a break in the circuit as I lower my pipette with retaining current, I withdraw and break the pipette tip (as this usually means there’s connective tissue in the way). I don’t proceed unless I reach my target with no interruptions in current. There’s no opportunity for a short, either (the red contact is not touching any metal other than the silver wire in my pipette).

Expression will often be pretty all-or-nothing, even within mice injected on the same day with the same batch of viruses. It’s been a nightmare to troubleshoot and I was wondering if anyone has seen anything like this while using the protocol.

Thanks!

2

Thank you for your question. Here is a response from a subject matter expert:

Thank you for the information regarding the Midgard Precision Current Source. We were not aware of this and will begin looking for a suitable replacement as well.

Regarding troubleshooting the issues you’ve been experiencing, there are a few tips and tricks we employ that are not expressly laid out in the protocol we provided. First and probably most obvious is to make sure that the wire itself is not corroded and is not itself blocking the pipette or shedding debris that could block the pipette, as this will not be clear from your voltage reading that a problem has occurred. Second, we always break off a small amount of the tip just prior to inserting into the brain to make sure virus has not coagulated on the tip. Third, we generally do not reuse pipettes if there are multiple injection sites. It’s typically one and done. Fourth, make sure at least about 2uL is loaded into the pipette. Low volumes correlate with poor expression. Beyond these, its important to note that we still expect a non-zero fail rate. We generally design studies with sufficient replicates that allow for 10-20% expression failures. Hope this helps and feel free to reach out with any additional questions or feedback!