Most PIs utilize the Mouse Embryonic Stem Cells Core to target and grow their ES Cells for microinjection. All requests for ES Cells microinjection are coordinated with the Mouse Embryonic Stem Cells Core. Please make sure to indicate in the note section of the request that the Mouse Embryonic Stem Cells Core has your ES Cells.
ESCI: For 129 ES cell injections the core uses C57BL/6J super ovulated blastocysts as donors and implant the injected blastocysts to ICR pseudo pregnant ICR females to get founder pups.
ABC57ESCI: For C57 ES cell injections, the core uses albino C57 blastocysts from natural mated females as donors and implant the ES-cell injected blastocysts to ICR pseudo pregnant ICR females to get founder pups.
For those targeting and growing the ES Cells please follow the protocol below. Note: All ES Cells must undergo murine viral testing before they can be injected. Proof must be sent to the GEM Core through email@example.com.
1. Feed the 6 cm plate 3 hours before using.
2. Wash with HBSS-Ca-Mg.
3. Repeat wash.
4. Add 1 ml of 0.25% trypsin to plate.
5. Incubate at 37°C for 5 minutes.
6. After trypsinization is complete, add 2 ml ES media.
7. Transfer the suspension to a 15 ml tube.
8. Using a transfer pipette, pipette up/down 20 times.
9. Centrifuge at 1000 RPM for 5 minutes.
10. Remove supernatant.
11. Resuspend cells in 3 ml of ES media.
12. Aliquot 1 ml to 1.5 ml tube for microinjection. Place this tube on ice.
13. Centrifuge the remaining 2 ml at 1000 RPM for 5 minutes.
14. Resuspend the cell suspension in 2 ml of 1 X freezing media.
15. Aliquot into freezing tubes and freeze for later use.
16. Take the ES cells that are on ice to the GEM Core R119 by 9 a.m. the morning of scheduled injection.