IACUC / LARC STANDARD PROCEDURES

PRODUCTION OF HEMOTOPOIETIC CHIMERAS
(Posted November 2006)

Description of procedure:

Bone marrow or fetal liver cells are isolated from euthanized donor mice. Bone marrow or fetal liver cells of a desired mouse strain alone or mixed at a 1:1 or 1:10 or other ratio with bone marrow or fetal liver cells from another mouse strain of interest are transferred into lethally irradiated recipient mice.

Irradiation of recipient mice is performed by using a cobalt irradiator within the barrier facility. Mice are placed restrained in a rotating pie-shaped holder (to limit mobility and insure equal irradiation), and then placed in the irradiator to deliver a dose of 350-600R. The exact dose that is optimal may vary with strain. For B6 mice irradiated with a cobalt irradiator the optimal dose for hematopoietic ablation is 550R. The mice will be in the irradiator 2-5 minutes. Irradiation is repeated 3 hrs later for a total dose of 700R - 1200R (split dose irradiation is used to limit the non-hematopoietic toxicity -- usually intestinal damage). Doses or irradiation protocols other than these will be noted as an exception to the standard procedure in the PI’s approved IACUC protocol. Mice will be monitored on the day after irradiation and 3-4 times a week for the first 2 weeks after the second treatment to assure there is no acute illness.

Irradiated mice are injected within 18h after the second irradiation via the tail vein with donor bone marrow (1 - 5 x 10e6) or fetal liver (0.5 - 5 x 10e6) cells in 200-400 ul of sterile phosphate buffered saline. Alternatively the cells may be injected retro-orbitally into anesthetized mice. Cell sources or numbers outside this range will be noted as an exception to the standard procedure in the PI’s approved IACUC protocol. Each irradiated mouse will receive a single intravenous (tail vein) or retro-orbital injection of donor bone marrow cells. Anesthesia is not required during the tail vein procedure, but is required during the retro-orbital procedure. If anesthesia is used, it should only be isoflurane since these mice will be debilitated.

Recipient mice are maintained on antibiotic-containing water or feed for 4 weeks. Duration of the entire procedure is typically 6-12 weeks (deviations to be noted in approved IACUC protocol).

Literature search words required:

Literature search was performed for refinement of this Standard Procedure on Sept 1, 2006 :

Key Words	Search Site	Years Covered
Irradiation (mouse)	Pubmed	1937-2006
Tail vein injection (mouse)	Pubmed	1961-2006
Restraint (mouse)	Pubmed	1957-2006

Agents:

• Isoflurane, bone marrow cells, therapeutic agents...
• All agents administered to animals should be listed in the “Agents” section of RIO.

Adverse Effects:

Adverse Effects
Procedure, Agent or Phenotype	Potential Adverse Effects	Management
Bone marrow transplantation	Transplant failure, anemia, infection, intestinal bleeding	Housed in barrier facility to prevent infection, if these effects are seen, animals will be euthanized

Monitoring Parameters
Monitoring Parameters	Frequency	PI/Lab will Document
Grooming habits, skin color, lethargy, general appearance/activity level	Day after irradiation, day after bone marrow injection and 3-4 times per week for the 2 weeks following irradiation. Once per week thereafter	No
Ability to eat and drink	Day after irradiation, day after bone marrow injection and 3-4 times per week for the 2 weeks following irradiation. Once per week thereafter	No
Body condition scoring	1 x on day of bone marrow injection and then 3-4 times per week for 2 weeks, once per week thereafter	Yes

Describe the conditions, complications, and criteria (e.g. uncontrolled infection, loss of more than 15% body weight, etc.) that would lead to removal of an animal from the study, and describe how this will be accomplished (e.g. stopping treatment, euthanasia).

Mice with Body Condition score <2 will be euthanized.

For all investigators housing animals with tumor formation, skin lesions, neurological deficits, or that are in Category E, list the expected characteristics/clinical presentations and endpoints of the animal model and the criteria for euthanasia. Note: The IACUC also requires such lists to be posted in the respective animal rooms and monitored by the IACUC compliance staff and LARC, to assure PI adherence to the endpoints listed.