Blastocyst injections:
Microinjection of ES cell lines into C57BL/6 or C57 Albino blastocysts and surgical implantation of injected blastocysts into pseudopregnant CD1 (or ICR) foster female mice.
The procedures required for making genetically altered mice are approved in the ARCH animal protocol; 11-04-1939R and the Harvard Committee on Microbiological Safety (COMS) license 01-105. However, the individual lines of mice generated by the IDDRC facility must be registered by the investigator with the Harvard COMS and approved in an animal protocol reviewed by the investigator’s institution’s animal care and use committee (IACUC).
Users need to arrange for the transfer of litters with chimeras during the week of postnatal day 7. ARCH will not approve transfer without proof that the animals are on the investigator’s animal protocol. Therefore animal protocol approval of the line is required before injection. If the animals are transferred after the week of P7, animal housing charges will be passed on to the user at twice the current ARCH housing rate.
For injection of “Outside” ES cell lines obtained from a source other than our Gene Manipulation Core:
-All cell lines need to be pathogen tested. Please use the Impact profile IV at the University of Missouri. or a similar service. Testing of “sister” lines that have been cultured in parallel to the injection lines is sufficient. (Most sources of ES cells have documentation of pathogen testing already)
-We strongly advise that all cell lines be karyotyped. Cell lines with good karyotypes may be given scheduling priority. The quality of the karyotype procedure and assessment must be confirmed by a member of the facility. A protocol for karyotyping is available upon request.
-For any given injection date, please plan on growing your cells according to our protocol (Web Prep of injection cells v.doc See Links and Resources) and bring us the single cell suspension by Noon on the scheduled injection date (usually a Friday). We can know on the Tuesday preceding the Friday injection how good a chance we have of being able to do two lines. Even if we feel we can only do one line, you may want to prepare multiple lines so that we can choose the line that looks the best.
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EXPECTED RESULTS
Blastocyst injections:
When using J1 cells that were targeted in the IDDRC facility and that have high karyotypes (90 –100%), a single round of injection frequently (90% of injections) results in the birth of pups with a high percentage of agouti coat color indicating good chimerism. About 90% of these lines will “go germ line.”
Cells that are unkaryotyped and/or produced in facilities other than the IDDRC have been more variable.