WHAT YOU WILL PROVIDE PART I
You will generate 5 females plugged on the same day, on a day agreed to in advance with us. Because the matings are done without hormone stimulation, you will need 10 stud males and a large cohort of 25 young, sexually mature females (6-8 weeks). If you can identify estrus females, you can use a smaller number of males. The males should be experienced in mating but rested for a few days prior to attempting these critical matings.
After coordinating with us, you will place 2-3 females with each male on the Sunday. On the Monday morning, you will check plugs. If one quarter of the females were in estrus, we would expect 5-6 females to have mated. Remove all the females and house the plugged females together. On Thursday, bring the plugged females up to your lab.
WHAT WE WILL PROVIDE PART I
On Thursday we will come to your lab and dissect out the uteri from the plugged females you generated above. We will transport the uteri to our lab to flush embryos. Our goal is to obtain 25-40 embryos total for the entire experiment. If enough are generated at the first attempt, then there will be no need for further breeding. However, if the yield is inadequate, another round of breeding will be required. We will inform you of the yield of embryos from the first attempt and of the need for further breeding. Normally the second attempt will occur the following week, in exactly the same fashion as above.
Once flushed, the embryos will be individually put into culture. After attaching to the feeder layer and hatching, the embryo explants will be dis-aggregated on day 6 of culture. The cultures will then be observed regularly for the presence of embryonic stem cells (around day 11 of culture). The cells will then be regularly passaged until enough cells are obtained to freeze. DNA from each line will be provided to you for genotyping. The entire process takes 6 to 8 weeks. The typical result is 7 to 10 cell lines for screening.
WHAT YOU WILL PROVIDE PART II
You will determine the genotypes of the cell lines by Southern blot and inform us of the results.
WHAT WE WILL PROVIDE PART II
We will expand up to four cell lines which you wish to pursue further. Additional expansions will be subject to additional fees.
We will freeze at least two vials from the expansion, and will provide you with one vial and retain the other(s) in our freezer for safe-keeping.
We will provide you with serum and instructions on how to thaw the cells and obtain an early passage pool of cells. At that point we expect you to be able to adapt the cells to grow them independently of us. We will provide further consultation as needed. Recovering cell lines from our backup vials will be subject to additional charges.
Four ES cell lines.