Detailed Project Information

ES Cell Injection

Information you need to provide prior to scheduling blastocyst microinjections:
Investigators must have an NUACUC approved animal protocol describing the use of animals generated by the TTML before microinjections can be scheduled.

• Investigators from CMRC must also provide a copy of their current Institutional Biosafety Committee approval letter
• A completed ES Cell Microinjection Request Form (Word)
• Evidence of rodent pathogen testing (MAP or equivalent PCR based analysis) of parental ES cell line
• Mycoplasma results on individual clones
• Chromosomal evaluation of targeted clones is also recommended

Once this information is received, we will schedule blastocyst microinjections.
Information regarding scheduled injections:

• 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 C57BL/6 blastocysts are injected and transferred into E2.5 surrogate females.

Expected results:

• 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

Project updates- we will notify you regarding:

• Scheduled injection date(s)
• Pregnancies and expected due dates
• Number of pups born and date of birth
• Number of chimeric pups, their gender, percent chimerism and ear tag number
• Transfer of pups to your animal room

Your responsibilities following injections:

1. Transfer of animals: once pups are weaned, TTML will initiate the paperwork for the transfer of these mice to your animal room and protocol. Your signature is required before CCM will transfer the mice.
2. 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.

Costs and Services