Transgenic Research Center In the Cancer Center

ES Cell Microinjection

Before we perform ES cell microinjection to generate chimeric mice, we require that (1) ES cells have a normal karyotype; (2) Pathogen free (see details below). We microinject two to four different ES cell clones for each knockout/knockin DNA construct.

For users who do their own ES cell manipulation procedures, all ES clones need to be thawed and passed for one generation prior to microinjection. On the day of microinjection, investigators should prepare the ES cells according to Protocol for preparing ES cells for microinjection.

To comply with current Stanford Veterinary Service Center guidelines regarding injection of cell lines into mice, the transgenic facility will send all ES cell lines from outside of our facility to the VSC for pathogen testing prior to blastocyst injections. The testing fee will be charged to the investigator. At the time of requesting of blastocyst injection, the user needs to give us a frozen vial of ES cells so that we can send the cells out for testing. We will then schedule the injection for you once we receive the testing results.

For microinjection of ES cells generated in our facility, we will expand and prepare the ES cells for users prior to microinjection. If the facility's ES cells (R1 and CGR) are used for gene targeting and handled by facility personnel, we will provide a minimum of four chimeric pups of high percentage chimerism and guatantee germline transmission. For other ES cell lines and ES cells handled outside the Facility, we inject up to 100 blastocysts per service fee and do not guarantee chimeras or germ line transmission.

Preparation of ES cells for microinjection

ES cells for injection should be in the log phase of growth. ES cells should be thawed and passed for one generation prior to microinjection. On the day of injection, the investigator needs to prepare ES cells by 9:00 am using the following procedure.

- Change media early in the morning and let cells grow in fresh ES media for 1 to 2 hours.
- Trypsinize ES cells into single cell form.
- Take trypsinized cells and avoid taking bottom-layer feeder cells.
- Wash trypsinized ES cells once with ES cell media.
- Resuspend cells in M2 media (obtained from the facility) at a concentration of 2-5 x 10^6/ml.
- Keep cells on ice.
- Give 1-2 ml of the ES cell suspension on ice to the facility located in room AF025 or AF027 in Research Animal Facility.

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