Injection Chimera

Prepare MES cells Remove media and wash in PBS once Add 1ml of Tryple E and incubate for about 10 minutes. Stop Tryple E action by adding MESC culture media containing FBS. Centrifuge cells 200g for 5 minutes. Pour off supernatant and resuspend in 1-2mls of MESC culture media. Prepare needles as needed for system. We will be using the Sutter Xenoworks systems and will demonstrate this in the laboratory. One pipet is for holding the blastocyst (inner diameter: 20-25mm) while the second pipet is for injection (beveled; approximately 1517 m internal diameter). Prepare an injection plate: In the LID of a 100mm Petri dish place two drops of 100ul of M2 media and one drop of the ESC culture media. Use the top M2 media drop to prep needles (remove excess bubbles). The middle drop for the embryos and to inject. The ESC media drop to place the ESCs after Tryple E digestion. Prepare embryos: Choose only fully expanded blastocysts to inject that are not hatching. Select five (the more experienced and efficient that you become this number can be increased) to transfer to the injection plate (middle drop). Inject: Choose small colonies of 5-10 cells that are fluorescent to aspirate into the pipet. Choose a blastocyst to inject and gently hold in the holding pipet with the ICM at 6 or 12 oclock. Bring the injecting needle close to the embryo and adjust it to the proper focal plane with the embryo. Gently touch the zona with the needle and then in a swift and steady motion release the pipet into the embryo. Inject cells into the blastocoel and remove pipet in a smooth constant motion.

The blastocyst will generally collapse after the injection pipette is removed but will expand in culture within a few hours. Return chimeric blastocyst to the incubator and hold for at least 1 hr before embryo transfer.