ES Cell Injection
The TTML can culture and/or inject ES cells for investigators who have either generated targeted clones in their lab or obtained ES cell clones from another source. Investigator provided ES cells are injected into the cavity of an expanded blastocyst stage embryo and injected embryos are surgically transferred into the reproductive tract of recipient females.
A C57BL/6 mouse colony is routinely available for microinjection of 129- and agouti B6-derived ES cells. An albino population of B6(Cg)-Tyrc-2J/J is also maintained in a more limited capacity for investigators with C57BL/6-derived ES cell clones. Investigators with ES cell lines derived from other strains should contact the laboratory for additional information.
Not all ES cell clones will result in the production of chimeric mice and not all chimeric mice are capable of transmitting the mutated allele though the germline. The ability to generate germline transmitting chimeras rests solely with the pluripotent capacity of the microinjected ES cells. Keeping ES cells in a non-differentiated state is largely a function of how carefully the cells are treated/handled during culture.
For these reasons, when working with ES cell clones cultured outside our lab, we cannot guarantee that microinjections will result in the generation of chimeric mice or that chimeric animals that are produced will be germline competent.
We have successfully generated germline competent chimeras from investigator-supplied targeted clones derived from the following parental ES cell lines:
- JM8.A1 (Agouti B6N)
- JM8.A3 (Agouti B6N)
- JM8.N4 (B6N)
- VGB6 (B6N)
- R1 (129X1 X 129S1)*
- G4 (129S6 X B6Ncr)*
- E14TG2a (129P2/OlaHsd)*
- AB2.2 (129S5/SvEvBrd)*
- GS1 (129X1/SvJ)*
- W9.5 (129Sv)
*Note that 129 substrains have complex genealogy and genetics; the major substrains have recently been given new nomenclature.
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 NUcore 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 (view 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- to 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.