Cryopreservation and storage of genetically altered mouse strains. Reanimation of cryopreserved strains via in vitro fertilization (IVF).
Spontaneous mutations constantly arise in every mouse colony. Some of these mutations may spread throughout the colony until all mice are homozygous for them. These mutations may affect the phenotype of the line. However, cryopreservation can greatly reduce the genetic drift’s impact.
The cryopreservation service preserves mouse lines by sperm freezing.
a) Sperm will be produced from client’s activated male and they will be frozen according to the well-established protocol.
b) At least 4 males of breeding age (3-8 months) of proven fertility are required to preserve a line.
c) At least 20 straws of sperm from three male mice are stored in two separate locations.
d) Quality control of frozen sperm is assessed by thawing one straw of frozen sperm for IVF, with wild-type oocytes. The resulting embryos are transferred to pseudo pregnant females to ensure they produce viable offspring.
e) Embryo tissue samples are provided to the client for PCR verification of the strain.
Cryopreservation
Cryopreservation of mouse strains serves a number of important functions:
•It provides reduction in number of mouse cages and reduction of associated maintenance costs.
•For strains that are difficult to replace, it provides insurance against disaster or pathogen outbreak.
•Cryopreservation provides insurance against genetic drift.
Genetic drift is not trivial. Spontaneous mutations constantly arise in every mouse colony. Some of these mutations may spread throughout the colony until all mice are homozygous for them. These mutations may affect the phenotype of the line. However, cryopreservation can greatly reduce genetic drift’s impact.
We strongly recommend that you cryopreserve any mouse strain that is critical to your research that either you created yourself, or that may be difficult to reacquire. If a breeding error, genetic drift, loss of phenotype, or a pathogen outbreak hits your colony, and you have the strain cryopreserved, you can recover the original strain and continue your studies with minimal loss of time.
The cryopreservation service preserves mouse lines by sperm freezing. Keep in mind that we use wild-type oocytes to thaw the cryopreserved sperm, and the result will always be heterozygote mice.
At least 20 straws of sperm from four male mice are stored in two separate locations (note: males are sacrificed for sperm collection). Quality control of frozen sperm is assessed by thawing one straw of frozen sperm for IVF, with wild-type oocytes. The resulting embryos are transferred to pseudo-pregnant females to ensure they produce viable offspring, at least till E14.5. Embryo tissue samples are provided to the researcher for PCR verification of the strain.
Procedure
In order to preserve a line, the following is required:
• Complete the Cryopreservation request form - frozen/thawed mouse sperm.
•Please be aware that the strain name entered on the request form will be the name used on the straw labels (maximum of 16 characters).
• Complete one request form for each strain required to be frozen.
•At least 4 males of breeding age (3-8 months) of proven fertility are required. For any mouse older than that, we cannot assure sperm quality and we will only perform the task after specific request from the P.I. and under their responsibility.
•A time table will be given. The researcher is responsible to hold each male in a separate cage with one female for 5 days for the renewal of the sperm cells, separate the males from the females and keep each mouse individually for 5 more days before sending the mice to the GEMM unit.
•When transferring the cages of male mice for freezing, the order number (which you will receive by e-mail) must be clearly written on the cage label.
In the case of complicated genotypes (more than one mutation), or if there is strain information that we should be aware of, please contact the unit management before initiating the order.
In the event of a waiting list for freezing, we will notify you, and we appreciate your patience and cooperation.