RRC-TPS: ES Cell Microinjection
- The investigator must open both an RRC account and a BRL account. The BRL account will be used to purchase and house the required mice. The investigator must also review our User Policies.
- The investigator must submit a UIC Animal Use Protocol for approval by the Animal Care Committee (ACC) before initiation of this project. When preparing your Protocol you should refer to the TPS ACC Protocol for generating chimeric mice (contact TPS Director). Any plans for experimental usage of mice subsequent to the generation of chimeras must be described in your Protocol application and approved by the committee.
- The investigator must complete the Application for ES Cell Microinjection (contact TPS Director).
- When the completed Application form is submitted to the TPS, the investigator and Director will discuss the necessary preparations and arrange a time for performing the microinjections. Mice will be ordered to arrive 1-week ahead of time. The TPS will take responsibility for keeping an accurate daily account of the number of mice being housed under the investigator's BRL account.
- On each day of microinjection the investigator will provide pathogen-free (mycoplasma testing of targeted clones is available at the DNAS facility; all parental ES cell lines must have been screened for a panel of mouse pathogens - contact TPS for details) ES cells in suspension at a scheduled time. For an additional fee, mycoplasma-free cells may be submitted as frozen stocks to be expanded and prepared for microinjection by the TPS. These cells will be submitted for additional pathogen screening, as necessary, for an aditional fee.
Generally, embryos will be microinjected with ES cells carrying a given targeted mutation over two (1-2 clones) or three (3-4 clones) days. A single targeted clone may be injected but we recommend that you provide more than one clone, as clones may vary with respect to their potential for germline transmission.
Approximately 8-10 injected embryos will be transferred into each of 4-5 pseudopregnant surrogate mothers on each day of injection, although variability with regard to these numbers should be expected (Note: occasional unforeseen problems may require that additional mice be ordered and injections carried out over a longer period of time).
- The investigator will be notified when the microinjections have been completed and when the potential litters are due (17-19 days after microinjection).
- At weaning (3 weeks) chimeric mice will be transferred out of the TPS to the care of the investigator. Prior to this time please make the appropriate arrangements with the BRL for housing your mice. The TPS and the BRL staff will be responsible for transporting the mice. Note, for some housing locations on campus mice must be screened for murine pathogens prior to the transfer. Fees associated with these tests will be charged to the investigator's BRL account.
At approximately 8 weeks of age chimeras are ready for mating with mice of the appropriate background strain for determination of germline transmission.
The number of chimeras produced and subsequent germline transmission is highly dependent on several factors, which include culture conditions, chromosome complement and acquisition of secondary mutations. We cannot guarantee success with every ES clone.
- Please acknowledge the Transgenic Production Service if you use mice produced in our facility in a publication.