1) Deposition mouse strains in the RIKEN BRC
In the RIKEN BRC, all the collected living mice are detained in a quarantine
facility equipped with disposable cages and filtered chambers. They are
then examined by serological tests to detect eight important infectious
microorganisms including lymphocytic choriomeningitis virus, mouse hepatitis
virus, and ectromelia virus. Frozen embryos and sperm are also received
for deposition of the mouse strains.
2) Clean up of deposited mouse strains
Based on the results of microbiological monitoring in quarantine, the mice
are transferred into Bio-bubble housing facilities under either negative
or positive pressure and bred until an appropriate number of mice for clean
up are produced. All strains are subjected to either in vitro fertilization or Caesarean section treatments. Diagnosed as Specific Pathogen
Free by the final quarantine tests using embryo-recipient or foster mothers,
the mice are transferred and maintained in the BRC main building. In special
cases, reproductive techniques such as ovary transfer or intracytoplasmic
sperm injection are used to clean up the strains.
3) Husbandry in the main SPF facility
The SPF facility in the main building of the RIKEN BRC has been planned
and constructed as a barrier facility for genetically engineered mice.
The air conditioning of each mouse room is regulated and maintained at
a constant temperature (24oC ± 2oC), relative humidity (55 ±10%) and ventilation rate (7 air changes/hr).
All the animal rooms and corridors are supplied with HEPA-filtered fresh
air with appropriate pressure to maintain the clean environment within
the barrier facility. The mouse rooms are illuminated at 300 lux (1 m above
the floor), with a light cycle of 12L/12D (08:00 on/20:00 off). Some strains
are supplemented with commercially available paper nests. All supplies,
including caging and water bottles, are autoclaved. Gamma-irradiated commercial
food is supplied. Cage cleaning is carried out by well-instructed animal
care technicians. These technicians maintain breeding records on cage cards
and files on each strain. The mouse racks, floors and walls are regularly
cleaned with a mild disinfectant solution. A card-key system safeguards
the animal facility and helps to protect against inadvertent microbiological
4) Cryopreservation of the strains
In the RIKEN BRC, the strains of mice that are frequently requested or
difficult to freeze-store are maintained as live animals. Most strains
are cryopreserved as embryos or germ cells whenever possible. The embryos
are produced through in vitro fertilization and cryopreserved at the 2-cell stage. Mutant strains and
genetically engineered strains are also stored as frozen sperms. Embryos
are frozen in 1.8 ml plastic cryotubes by vitrification with a low-toxicity
EFS (ethylene glycol, ficoll and sucrose) solution
Kasai et al., 1990, J. Reprod. Fertil., 89: 91-97) or DAP213 (DMSO, acetamide and propylene glycol) as a cryoprotectant
Nakao et al., 1997, Exp. Anim., 46:231-234). Mouse sperms are collected from the cauda epididymis, suspended in an
18% raffinose and 3% skim milk solution (
Nakagata, 2000, Mammalian Genome, 11: 572-576), embedded in plastic straws and freeze-stored in liquid nitrogen. ES
cells of genetically engineered mice are also frozen-stored.
5) Maintenance and preservation of mutant strains requiring special care
In the RIKEN BRC, mutant strains that are difficult to breed and require
special care are also maintained and preserved with the aid of reproductive
technologies, such as in vitro fertilization, ovary transfer and intracytoplasmic sperm injection.