Prior to blastocyst injections
Investigators must have an approved animal protocol describing the use of animals generated by the TTML before microinjections can be scheduled.
- Complete the request form found on the NU Core TTML page and upload when ready to purchase on NUCore.
- Evidence that parental line has recently tested for infectious pathogens (recommended IMPACT profile)
- Mycoplasma results on individual clones
- Chromosomal evaluation of targeted clones is also recommended
Preparation of ES cells for blastocyst microinjection:
- Investigators have the option of having the TTML prepare cells for microinjections or preparing the cells themselves.
- Investigators preparing cells in their lab should prepare cells for microinjection in injection media (ES cell growth media freshly supplemented with 20mM HEPES) and bring both the prepared cells and 5 ml of injection buffer on ice to the laboratory by 12:30pm on the scheduled injection day. (Protocol).
- Please note: For investigators on the Chicago campus, it is very helpful to us to observe cells in culture prior to trypsinization on injection day, if possible, so that we can assess the relative health, density and degree of differentiation of the clone scheduled for injection.
- If the TTML is preparing the cells, we will need a vial of frozen cells, growth media specifications, and information on the parental ES cell line including its growth characteristics, if known.
Blastocyst microinjections: a minimum of 36 E3.5 blastocysts are injected and transferred into E2.5 surrogate females.
- All chimeric animals generated will be provided.
- We cannot guarantee the generation of chimeric pups from clones provided for injection as there is no way of knowing how cells were grown or handled by others prior to arriving in the lab.
- We will make recommendations based on the appearance or growth characteristics of clones we observe in culture.
Timeline for scheduled blastocyst injections
- Week 1: order embryo donor mice
- Week 2: mice arrive; preparation of donor and recipient females
- Week 3: preparation of ES cells and blastocyst microinjection
- Week 6: pups born 17 days following microinjection
- Week 9: 3-4 week old chimeric pups weaned and transferred to PI
Your responsibilities following injections
- Transfer of animals: once pups are weaned, TTML will initiate the online transfer of these mice to your animal room and protocol.
- Breeding of founders: breeding and subsequent experiments are, of course, your responsibility. Our staff can provide consultation on animal handling, breeding and housing, if necessary.