The GEMF is now able to generate novel ES cell lines from blastocysts. This technique will enable the investigator to obtain ES cells from their unique strain of mice for further genetic manipulation or in-house testing.
We will superovulate five to 10 wild-type or homozygous females (supplied by the PI) and mate them to males supplied by the investigator. Blastocysts will be isolated and grown to obtain the inner cell mass. The ICM will then be dissociated to obtain ES cells. ES cell lines are typically generated from about 10% of the blastocysts isolated. Depending on the experiment, these lines can be sent for cytology and then injected (for an extra fee) to determine germline transmission capability. We cannot guarantee the success of obtaining germline transmission. However, we will work with the investigator to achieve positive results to the best of our ability.
$5,000 for MD Anderson investigators; $9,450 for MD Anderson-affiliated investigators; $15,120/construct for other outside investigators
Time Line for ES Cell Generation
- Day 1: Treat females (wild-type supplied by GEMF, homozygous supplied by PI) with PMS
- Day 3: Treat the same females with HCG and mate to males supplied by the PI
- Day 7: Females are brought to the GEMF Cryo Lab by the PI staff. Blastocysts are flushed from the uterus of the females and plated on 6-cm tissue culture plates previously seeded with feeder cells
- Day 12: Blastocyst outgrowth is checked for the presence of a distinct inner cell mass (ICM). If a defined ICM is detected, it is carefully dissected out, lightly trypsinized and plated in a well of a 96-well plate previously seeded with feeder cells
- For the next four to eight weeks: Each well is carefully monitored for growth of ES cell colonies. The wells are trypsinized and moved to larger plates for growth as necessary. In addition, we will add 2i and 3i preparations (Li P et al. 2008 Cell 135:1299-1310; Ying QL et al. 2008 Nature 453:519-523) as necessary to encourage growth of the ES cells
- When the ES cells are growing consistently on a 6-cm plate, we will freeze down four vials of the cells (typically at passages 4-6). We will supply the PI with an aliquot of cells to verify the genetic composition, and can supply a plate of cells for cytogenetic analysis
- We will also inject the ES cells into blastocysts (for an extra fee) if the PI wishes to establish that the cells are capable of germline transmission