Virus Stuff - theunissenlab/lab-documentation GitHub Wiki
Spreadsheet of our viruses and tracers: https://docs.google.com/spreadsheets/d/1IoHLA1Vx5mFDU2Qdr84U-NRE-BnXGhTsIZTp1CRBKiQ/edit?usp=sharing This same spreadsheet is where we keep records of training for our BUA.
We almost only work with AAVs without dangerous transgenes, they are considered harmless and thus are BSL 1. We are approved to use Cav-2, which is BSL2. Thus our lab is certified as BSL2, which is why we have a BUA.
We do have annual inspections. Chips (Annika as of 2023) is the lady to talk to, she is very nice. https://ehs.berkeley.edu/biosafety/how-do-i-get-or-renew-bua
Everyone working with viruses needs to be sure to take EHS 201: Biosafety in Laboratories. Go to https://jwas.ehs.berkeley.edu/lmsi, then search for "biosafety".
Everyone also needs to be trained by the PI in our particular protocols. This involves being given a really stellar PPT here.
You also need to read our BUA, here. The newest document is from 2024. They are moving to an online system but as of Feb 2024 haven't gotten it entirely online yet.
More information on BUA inspections is available on the inspections page (here)
Clean-up Procedures
We have a sharps container dedicated to our AAV work. When it is full, or 30 days after it was first started, it should be disposed of. You can use any sharps container that has a biohazard sticker, including the ones in the surgical suites.
Pretty much any waste generated during a surgery involving AAV needs to go into biohazard. This includes gloves and other PPE. This trash is then placed in the biohazard bags in the cold room (code 123456) where they will be autoclaved by staff. This trash can be either a red biohazard bag (in which case the trash can must have a lid) or a white autoclaveable bag (in which case the trash can does not have to have a lid). It's kind of a weird policy.
If you generate waste that's not supposed to go into the sharps container but is small (like an eppendorf tube) you can either take it directly to the cold room biohazard bag or you can store it as "accumulation" in a white bag until you want to take it upstairs. "Accumulation" is when something isn't quite trash- this is straight from Chips, our BUA officer.
In the instance of a spill, we will use PREempt Hydrogen Peroxide Disinfectant Wipes as our primary decontaminant. These are available to buy in the Barker Storeroom. All spills should be reported to EH&S. We also have a spill kit in 123D, and the bleach needs to be checked yearly because it expires.
Note that alcohol is apparently not an effective means of cleaning up viruses.
Bedding
For the first 72 hours post-injection there is a chance the animal may shed virus. During this time the bedding must be autoclaved. Our protocol is to change the bedding 72 hours after surgery, place it in a red biohazard bag and place that in the red biohazard bag in the cold room. After 72 hours the bedding can be disposed of normally.
Safety Protocols during use
Wear closed-toed shoes, lab coat, and gloves. If you are pipetting the virus (aliquoting, for instance), there is a chance of aerolizing thus a chance of an inhalation hazard, and this should be done in the back of the fume hood and a mask should be worn, probably with eye protection for good measure.
During surgery we inject the virus via a puff of air, which means there is a risk of it splashing upwards into your eyes. Wear eye protection and a mask.
Random Notes Bill made a while ago about our first virus:
The plasmid: https://www.addgene.org/107790/
Penn vector core. the virus is AAV9.CamKII.GCaMP6s.WPRE. Vector: AAV Capsid Serotype: 9 Promoter: CamKII Transgene Sequence: GCaMP6s, WPRE
Notes about the serotype and the virus construct in general: Will Liberti seems to think it will work, hopefully it does.
Notes about the promoter: Zebra finches appear to have three CamKII’s, who knows which ones will activate our promoter, hopefully all of them. Importantly (and interestingly), all CamKII genes appear to be down-regulated in HVC, RA, and lMAN. The auditory system generally looks to have normal expression. Field L seems to be down-regulated. CamKK may be upregulated in HVC and RA. http://www.zebrafinchatlas.org/gene_search?pattern=camk
Notes about the calcium indicator, GCaMP6s: The ‘6’ indicates the currently (2018) most advanced flourescent calcium indicator we can get. The Adesnik lab may have something better, and we may want to talk to them eventually. The ‘s’ is the brightest, but not the fastest, version of this protein, see Chen et al 2013. Ultrasensitive fluorescent proteins for imaging neuronal activity.
Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) is a DNA sequence that, when transcribed creates a tertiary structure enhancing expression. Commonly used in molecular biology to increase expression of genes delivered by viral vectors. WPRE is a tripartite regulatory element with gamma, alpha, and beta components.
Carmena lab viruses: https://docs.google.com/spreadsheets/d/1eojqDOVwsKMuUKZFZmcW4Tlz4lTBo5TZUbnJ0nz2eME/edit#gid=0