We got our construct packaged into AAV particles from a vector core at a reputed institution. We received 10x 100uL aliquots (all made in the same prep).
I further aliquoted 1 of them into 5uL aliquots in December 2016 stored at -80C. We used all 20. No problem.
I thawed out a new one in September 2017, used 3 aliquots had no issues either. A rotation student in the lab injected some mice with 1 aliquots in October 2017, and the mice seemed lethargic after, almost moribund but they recovered. We just blamed it on their inexperience and moved on.
Now, a post-doc in the lab injected some mice with the 6th aliquot , and all of his mice died. I injected 2 mice - 1 with a fresh 5uL aliquot (6th aliquot) from -80C and 1 with another virus that was aliquoted around the same time as this one. Same bag of tubes used to make the aliquots. Stored at -80C.
The first mouse died and the other survived.
Injected a few different mouse lines we have - they all died.
I thawed out a new 100uL aliquot and aliquoted it into 20 x 5uL aliquots. Now I injected 2 mice with the new aliquot and 2 with a new 5uL aliquot of the old (7th aliquot).
The old virus killed both mice within hours of surgery. Mice injected with the newly aliquoted virus survived and were doing very well.
The mice woke up from the surgery, seemed to have groomed off the eye ointment, but were just laying down. One of the mice had a foamy white eye discharge before death.
My site of injection is under the lateral ventricle. So normally when I pull my syringe out, I get some CSF leak. It is clear looking. However, the mice injected with the "old virus" had bloody, cloudy CSF come up.
It does not make sense to me that something developed in my aliquoted virus over the period of a month (AT -80C!) that is causing this? And if thats the case, then why that 100uL aliquot and not the others?
Anyone have any guesses?
Maybe you are hitting a major blood vessel on the way...how big is the syringe you using and how fast what volume are you injecting? Did you change anesthetics - fresh bottle?
Are they all dying post surgery (hours within?), or also on the operating table? Hard to imagine it's a virus problem - you could maybe try in a cell culture to see if there is toxicity problem with the viral batch.
Hi Michael, I did all the surgeries in the same sitting , injecting with the same coordinates and the same kind of syringe. But only the ones with the old virus had the bleed and the new ones were ok. I staggered the injections as old - new to avoid any issues with perhaps changes in anesthetic delivery over time.
They all woke up from surgery but only the ones injected with the old virus died after.
It makes no sense to me either that it could be the virus itself or the viral prep. If it was a problem with the viral batch it should have given me a problem when i used the first 100uL of it, but it didn't. All the 10 100uL tubes come from the same batch and prep. But Yes I'll test in culture! That's one thing I haven't tried yet . Thanks!!
Hi Raj,
Do you have the same titre in all of your aliquots? The blood in the CSF might suggest that you are hitting a blood vessel as Michal said although it doesn't sound like you're seeing a lot of blood as you would expect in that case. Perhaps you could try injecting into a more superficial, less vessel dense region? If you do that you could use an old aliquot in one hemisphere and a new aliquot in the other and compare them in the same mouse.
Good luck!
The titter is the same. It is the same prep aliquoted into 100uL aliquots.
I am injecting the same region with the old vs. new aliquot. Only the old one caused bleeding in 3 mice whereas the other 3 mice had no bleeding.
I could try the same mouse thing!
No. I just didn't get a virus packaged from that vector core again. I switched to UNC.
get new tubes from a different supplier for holding the aliquots. Might be something in the plastic that leaches over time