Generation of Primary Mouse ES Cell Lines
Investigators must receive approval from the director of the core for a project to be initiated.
Complete and submit the Generation of Primary Mouse ES Cells Form.
- Complete name of mutation
- Genetic background of parental animal
- Come to the core and pick up hormones if necessary
- Follow the instructions on the Super Ovulation and Plugging Schedule
- Bring plugged females to the core the morning of embryo harvest
The core will provide to investigator the hormones for super ovulation of female mice. Each investigator is responsible for administering the hormones using the Super Ovulation and Plugging Schedule. Investigator will bring the plugged females to the core on the day of embryo harvest. The core will flush and harvest blastocysts and prepare them for primary ES cell culture. A total of three cell lines will be generated from three independent embryos. Once culture of a line is established colonies will be plated to perform Alkaline Phosphatase staining. The enzyme Alkaline Phosphatase is present on the cell surface of embryonic stem cells. It is a useful tool which helps identify these cells. Investigator will receive a total of two vials at passage 5, five vials at passage 7 and 15 vials at passage 9 for each cell line generated.
After contacting the director of the core for project approval investigator must complete and submit the Generation of Primary Mouse ES Cells Form. The core supervisor will e-mail the contact person within three working days after the form is submitted to set-up a schedule and provide the hormones. Every request will be taken on a first come, first serve basis.
Investigator and or their staff perform hormone injections and plugging as stated on the Super Ovulation and Plugging Schedule.
On the day the core receives the plugged females embryos will be harvested and seeded for cell line generation.
First dissociation of embryos will take place 5-6 days after seeding embryos (p=1).
Colonies will be picked approximately 7 days after 1st dissociation and placed in 96 well plates (p=2).
Culture will be expanded into 24 well plates approximately 3-4 days after p=2 (p=3).
Culture expansion into 6 well plates will take place approximately 3-4 days after p=3 (p=4).
Culture will be frozen into 3 vials and re-seeded into 6 well plate approximately 3-4 days after p=4 (p=5).
Culture expansion to 100mm plate will take place approximately 3-4 days after p=5 (p=6).
Culture will be frozen into 5 vials and re-seeded into one 100mm plate approximately 3-4 days after p=6 (p=7).
Culture expansion into three 100mm plates will take place approximately 3-4 days after p=7 (p=8)
Cell line will be frozen into a total of 15 vials at p=9 approximately 3-4 days after p=8 (p=9).
Cells will be seeded for colony formation and Alk-phos staining will be performed 5 days later. Investigators will receive a photograph of the results of the assay along with the cell lines.
Therefore, investigators should receive any established cell lines approximately 40 days from the day they bring the plugged females to the core. Alk-phos staining results will be ready 5 days later.
In the event that the culture becomes contaminated, the core will immediately re-schedule the project at no additional cost to the investigator.
The director of the core will address other problems on a case by case basis.
After service agreement:
We would appreciate, that any publications arising from the generation of cell lines by this core carry an appropriate acknowledgment of this service.