Standard Operating Procedure

Intrathymic infection of hCAR mice with Adenovirus

Hazards:

ADENOVIRUS is a human pathogen associated with the common cold, which causes respiratory, ocular and intestinal infections. The modified virus we use is replication defective and cannot mount a persistent infection. Nonetheless, replication-deficient adenovirus may theoretically recombine with the wild-type virus to result in a replication-competent virus. Because the wild-type virus is very common, this can be a concern, depending on the nature of the gene being introduced. In our case the genes being introduced encode short inhibitory RNAs that are complementary to murine genes involved in thymic development. These genes are not inflammatory, regulatory, or toxic in humans as far as we know.

Ketamine and Buprenorphine are controlled substances and anyone working with these needs to take the training session and be registered with the SBOP (State Board of Pharmacy).

EXPOSURE TO ANIMALS: Personnel involved with animals may be exposed to bites, scratches, infectious agents, and allergens. Tetanous is a rare but potentially fatal bacterial infection possible after any animal bite. See also the Boynton Health Service brochures on animal bites and allergies.

CARBON DIOXIDE: toxic gas, avoid inhaling.

SHARPS: take appropriate cautions when working with needles

Protection:

Adenovirus: The handling of virus, infected cells, plasticware and liquids must only be undertaken by trained individuals. Lab goat, gloves, and protective eyewear will be worn at all times. For the surgery, wear a facemask as well. Whenever possible, work will be performed in a Class II flow hood. A biological hazard sign indicating the use of adenovirus will be placed on the biological safety cabinet and on all mouse cages.

Mice general: Wear a facemask if you’re allergic to mice or to prevent the onset of allergies. Have current tetanus immunization. If biten by a mouse, immediately wash wound with soap and water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).

Waste:

Infectious waste and potentially contaminated surfaces will be immersed in 10% bleach (dry, solid waste – pipettes, pipette tips, centrifuge tubes and caps, etc.) or mixed 1:1 with 20% bleach (liquid waste). After 30 minutes in bleach, the waste will be discarded in the appropriate standard lab biohazard waste boxes (deemed biohazard because of the bleach and/or sharps).

Carcasses of infected animals will be double bagged, frozen, and brought to MCB144A where there is a biohazard disposal freezer. RAR staff will ultimately incinerate them. Cages, including bedding, will be wrapped in autoclave bags and brought to the MCB144A for appropriate disposal and cleaning by trained RAR staff using appropriate precautions.

Spill Clean-Up:

Remove any contaminated clothing and put in red autoclavable biohazard bag.

Notify other workers in the area of the spill and control traffic through area.

Wear shoe covers if spill is on floor. Put on gloves and cover spill area with paper towels.

Pour bleach over towels from edges of spill to center, be careful not to splatter. Decontaminate all objects in spill area. Allow 20-30 minutes of contact time. Pick up any sharps, including broken glass, with forceps and place in sharps container. Wipe area with disinfectant and clean towels, mop if spill on floor. Remove gloves and foot covers before leaving area of the spill, put in biohazard bag, and wash hands. Autoclave waste.

Procedure:

Prepare reagents:

-Prepare a solution of the recombinant adenovirus (1x10e6 IU/ul) and transfer to a .3 cc tuberculin syring (29g needle).

-Ketamine xylazine mix: load 120ul/mouse into a 1cc syringe

-Buprenorphine: dilute stock 1:10 in PBS, load 30ul/mouse into a 0.3 cc syringe

-Have handy: Eye moisture salve, 70% EtOH, instruments (2 fine curved forceps, 1 fine scissors, 1 small scissors, cotton swabs), stapler

Prepare the surgery area: Wipe down the hood with 70% EtOH. Prepare an animal restraint device as shown in the diagram below using rubber bands, pins, and Styrofoam.

NOTE: Intrathymic injection surgeries are typically performed in the horizontal laminar flow hood in 6-250 NHH. For this procedure, you may begin the surgery setup in this hood with the flow on. However, as you approach the point of adenovirus injection, you must shut the flow off. Leave off for the duration of the surgery (skin staple and removal from restraint device).

1. Anesthesia: Inject animals with 80ul ketamine/xylazine mix i.p. (use 1 cc tuberculin syringe, with 28g needle) Note: This anesthetic is a controlled substance. The lab stock is kept in a locked box under Jie’s bench. The precise amount used should be recorded in the Disposition Log in the Controlled substances notebook above Jie’s desk. We are approved to inject 100ul of this i.p., which is 90 mg/kg ketamine for the average mouse (25 g). Use 80ul to start. If after 5 minutes, the animal is still not completely under, then a small amount more can be injected.

2. Surgery:

Apply eye moisture salve to the eyes of each animal to prevent them from drying out during the anesthesia period. Place into an immobilization apparatus made with rubber bands (see diagram, left).

Swab the chest area with 70% EtOH. Starting at the base of the sternum, make an incision through the skin, straight up to about the base of the chin. Press skin away from centerline using cotton tipped swab.

Using a clean fine scissors, start at the sternal notch, and make a 3-5 mm incision down through the sternum, exactly at the centerline. Using two curved forceps, gently open the ribcage, removing any tissue that may be directly above the thymus. The tip of the thymus should be visible. If not, you may need to make the incision longer.

Inject 10ul of a solution of recombinant adenovirus (total of 1x10e7 IU). Use a .3 cc tuberculin syringe, with a 29 g needle. When using a tuberculin syringe, it is easy to inject too much, so load only the 10ul.

Using a curved forceps hold the two flaps of skin together and apply 2-3 staples. Inject the analgesic buprenorphine (Buprenex). Dilute a .3mg/ml stock 1:10 in PBS, inject 30 ul of this (900ug) i.p.. Lay the animal on its side in a clean cage on a heating pad sent on low to recover (may take up to 2 hours for complete recovery). Label cage cards with “Biohazard – Adenovirus”

NOTE: be sure to soak all surgery waste (immobilization apparatus, rubber bands, cotton swabs, tubes, syringes etc.) in 10% bleach prior to disposal in an appropriately labeled biohazard waste container. Clean surgical instruments with bleach for 30 minutes and rinse extensively, before putting back in the instrument pool.

3. Analysis

Harvest thymus, spleen and lymph nodes. Stain for CD4, CD8, and any other markers needed. After infection in vivo, no infectious virus should remain or be produced. Therefore, the cells can be handled as usual.