Download Working with VSV-G PSEUDOTYPED MSCV

Goldstein Lab
STANDARD OPERATING PROCEDURES FOR VSV-G PSEUDOTYPED MSCV-RETROVIRUS
CARRYING BCR-ABL WORK IN HES ROOM
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Title
12
Tissue Culture (TC) Room, Leichtag Building Room #471
All
11/15/2007
1.0
Working with VSV-G pseudotyped MSCV-retrovirus carrying BCR-ABL in TC Room
Purpose:
Guidelines to provide a clean and safe TC environment and to maximize containment
and minimize potential for contamination with this oncogenic retrovirus.
1. Policy:
• Room #471 (TC Room) is a BSL2+ room. This means that we have permission to work there
with agents that need special treatment and safety practices (in terms of their maintenance,
handling and disposal). These agents include lentiviral vectors (BSL2), adenovirus (BSL2+), and
VSV-G pseudotyped retroviruses(BSL2-BSL2+). Every researcher using the TC room is
responsible for the maintenance of a clean and tidy, as well as a safe TC room. Please maintain a
good level of cooperation and communication.
2. Safety Clothing and Ante-room Issues:
• The outside door that leads into the ante-room is to remain CLOSED AND LOCKED at all times.
•The main door to room 471 is a “GLOVE OFF” DOOR.
• Both the outside and inside of THE ANTI-ROOM door will be a “glove on” door, i.e., ALWAYS
use gloves while touching the Anti-Room door handles. However change gloves if there is the
possibility of contamination on gloves before touching any door or equipment. Place a sign on
outside of anti room door letting others know you are working with LENTI or other VIRUSES. A
specific sign will also be posted saying BCR-ABL work is ongoing which will currently always refer
to this VSV-G pseudotyped MSCV-retrovirus. Only the already packaged virus will be used in this
facility as packaging has been done and will always be done at the Viral Vector Core.
• Personnel working with this particular retrovirus carrying BCR-ABL will gown with booties, double
glove, Disposable labcoat and protective eye wear. (If you are in the TC room while some one else
is using this retrovirus carrying BCR-ABL you too must follow these rules. It is advised to avoid the
room during use of this virus as it is particularly dangerous. However, times of use will always be
given ahead of time so arrangements can be made to work with or around peoples schedules.)
• When you are done with your experiment or leaving the main TC room, personnel working with
this retrovirus will discard gloves, labcoat, and booties in the main TC room biohazardous bags•
3. Working in TC room (BSL-2+ Area):
• The hood farthest away from the door (the right hood) is designated for BSL2+ work This means
that anybody working in the TC room (with or without BSL2+ agents) can work in the right hood,
but BSL2+ work should only be performed in the right hood.
• You MUST sign log sheets when using virus in either right biohood or bottom left incubator.
• All BSL2+ cell culture dishes will be maintained in the lower left incubator ONLY No other cells
can be placed in that incubator.
• BSL2+ virus and cells will always be inside a secondary container when outside of the hood. This
means when using the microscope, incubator, centrifuge, or carrying these cells between these
locations they will always be inside a secondary container during and 72hrs after transduction (for
example a larger plate). Secondary container will be placed on top of the small safety trays inside
BSL2+ incubator. The tray should be cleaned with 10% bleach 15minutes, wipe with 70% ETOH
and autoclaved after you have completed using that incubator or if a spill occurs.
• All BSL2+ cell culture and any virus infected cell will be maintained in the lower left incubator
ONLY during exposure to virus and within 72hrs post transduction. The incubator is label on the
outside “BSL2+ .” After this 72hrs post transduction has passed, cells can be maintained in the
other three incubators.
4. Working in the Right (BSL2+) Hood:
• Take an empty liquid container (Like a old PBS bottle) and a 50 ml conical and add a large
amount of 10% bleach to it.
• Place bleach (10%) containers labeled “Bleach for BSL2+ Work“ in the hood at the beginning of
your work and keep it there until you are finished.
• Place a double-bagged, red biohazard bag inside the hood for disposing all solid waste inside the
hood during the use of virus.
• Turn sign indicating virus work is in progress and post an additional sign that says “BCR-ABL
virus work in progress.”
• Bring virus into the room or begin working with virus.
• Do not use glassware.
• When using plastic pipets, SUCK-UP bleach from your 10% bleach containers into plastic pipets
every time before discarding them inside the hood into a red Biohazard bag.
• If using pipet tips, use “filtered” ones and SUCK-UP bleach from your 10% bleach containers
every time before discarding them inside the red biohazard bag inside the hood.
• Place a spray bottle with 70% ethanol inside the hood during the duration of the virus work to
spray everything off routinely, including gloves.
• Spray gloves with 70% ethanol routinely and every time you need to reach outside the BSL2+
hood to pick up other things/reagents in the TC room (eg. to pick up a bottle from the 37˚C
waterbath, etc.). Alternatively, change gloves if you suspect you got something on them and place
outer gloves inside the biohazard bag inside the hood.
• In the case of a small spill inside the hood, clean BSL2+ hood thoroughly with 70% ethanol.
Discard Kimwipes used for decontamination into biohazard bag inside the hood. DO NOT USE
10% BLEACH IN HOODS AS THIS WILL CORRODE THEM.
• Pipet ALL liquid BSL2+ waste into a disposable container with 10% Bleach. NEVER use
VACUUM with BSL2+ or possible virus containing liquid. When you are through with the
liquid waste be sure the total volume of the container equals 10% bleach. Error on the side of
extra. Be sure the bleach is mixed with the waste and allow 40 minutes inside the hood before
discard waste down the drain. Dispose of container in biohazard trash inside the hood. DO NOT
LEAVE LIQUID WASTE SITTING IN HOOD FOR LONGER THAN 30 MINUTES! If liquid is less
than 30mls total, dispose of whole container inside biohazard bag inside the hood to be removed
with other solid waste.
• When you are completely done you must double bag all disposable biohazard waste (pipets, tips,
gloves and any other biohazard trash) INSIDE the hood. Seal this bag inside the hood and place
inside the larger biohazard bag outside the hood.
• Clean BSL2+ hood thoroughly after removing biohazard bag with 5% LPH, allowed to dry, wipe
down with water to remove LPH film, and then wipe down with 70% ethanol after every use with
this virus. Place all cleaning waste inside big biohazard bag outside of hood.
• De-gown and replace outside gloves throwing all inside biohazard bag. Dispose in autoclave
room as soon as you are done!
• When this final step is completed and the entire room is decontaminated remove the sign
indicating “BCR-ABL work in progress.”
I have read the above document:
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