Chimaera production via blastocyst injection
The facility produces chimaeras by injecting into blastocysts ES cells provided by the investigator.
ES cells may be prepared either by the investigator, an external unit or the facility itself following a successful targeting.
• ES cells targeted by the facility
The targeted EScells are recovered and expanded from the frozen stock.
Depending on the genetic background of the ES cells the host blastocysts might be C57 Bl/6 (for 129ola or 129sv cells) or C57 Bl/6 albino (for C57 Bl/6 cells).
Female donor mice are superovulated and mated with fertile males. 8-cell stage embryos are recovered on day 2.5, and they are cultured overnight. The following day the grown blastocysts are injected with the targeted ESC. Injected blastocysts are transferred the same day in pseudopregnant foster mothers.
The pups will be born after 17-20 days, depending on the stage of the foster mother. Chimaeric mice along with their litter mates are delivered after weaning, at approximately 5 weeks of age. Breeding of the chimaeras to test for germline transmission is the investigator’s responsibility, although advice can be provided by members of the staff of the facility. In case it is requested the facility to do this step, then space and time permitting the facility can take over with an added cost.
Germline transmission of the chimaeras is guaranteed at the level of a single chimaera transmittion minimum.
• ES cells not targeted by the facility
When the targeted ES cells are provided by the investigator, they must be accompanied by proof that they are tested for mycoplasma. If this is not available, then the facility will do the test before using them.
The procedure we follow is the same as for the ES cells targeted by the facility. Germline transmission of the chimaeras can not be guaranteed, because is highly dependent on the quality of the ES cells provided.
|
|
To order please fill out the Blastocyst Injection Form
|
|
|
|
