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IACUC / LARC STANDARD PROCEDURES
PRODUCTION OF HEMOTOPOIETIC CHIMERAS
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 will be collected. These will be delivered alone or mixed at a 1:1, 1:10, or other desired ratio, with bone marrow or fetal liver cells from another mouse strain of interest and are then transferred into lethally irradiated recipient mice.
Irradiation of recipient mice is performed by using a cobalt irradiator within one of the barrier facilities. Mice are placed into a rotating pie-shaped holder (to limit mobility and insure equal irradiation), which is then secured 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). Mice will be monitored as described below to assure there is no acute illness.
Doses or irradiation protocols other than these will be noted as an exception to the standard procedure in the PI’s approved IACUC protocol.
Irradiated mice are injected within 18h after the second irradiation usually 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. Use of inhalant anesthetics (isoflurane) is recommended as these mice will be debilitated.
Recipient mice are maintained on antibiotic-containing water or feed for approximately 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 23, 2009:
| Key Words |
Search Site |
Years Covered |
| Irradiation, production of hematopoietic chimeras, tail vein injection (mouse) |
Pubmed |
1937-2009 |
| Irradiation, production of hematopoietic chimeras, tail vein injection (mouse) |
Google |
No limit |
Agents:
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 of less than or equal to 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. |
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