A targeting vector, provided by the investigator, will be introduced into ES cells via electroporation, and the electroporated ES cells will then be grown in appropriate selection media. ES cell DNA from individual clones will be given to the investigator for PCR or Southern Blot analysis. Positive ES clones will be expanded, frozen, and maintained in the facility for subsequent blastocyst injection to generate chimeric mice.
Time line for mouse ES cell gene targeting:
Day 1 |
Day 5 |
Day 12 |
Day 21 |
Day 30 |
Day 35 → |
|
| Start ES cell Culture |
Electrophoesis |
Pick up clones |
Extract ES cell DNA |
Expand positive clones |
Freeze cells in liquid nitrogen |
Blastocyst injection |
Factors to consider when designing a targeting construct:
- It is very important to use genomic DNA that is isogenic with the ES cell line to insure high levels of homologous recombination. Our facility presently has the129/Sv ES cell line available to investigators.
- One of the most important parts in designing a targeting vector is to identify a scheme to determine targeted clones. Screening can be conducted by either Southern blot or PCR. The probe for Southern blot analysis must be located outside the region targeted in order to distinguish between homologous and non-homologous recombination.
To request service from Transgenic Mouse Facility:
- Schedule an appointment with Fuming Pan (244-0649, fpan@uiuc.edu) to discuss the targeting strategy.
- Provide the facility with the DNA construct approval date from the Biological Safety Committee. The facility will not initiate any experiment without relevant approval date.
- Complete and submit the Service Request From for ES cell targeting, and provide at least 200ug linearized DNA at 0.5ug/ul or higher to the facility.
- For targeted ES cell selection, the cost is $4000. Fee is subject to change.
- The investigator will be responsible for the analysis of ES cell clones by either PCR or Southern blot, and convey the results to the facility.
About TMF Services & Fees Request Form Transgenic Gene Targeting Knockout Cryopreservation Rederivation
Mouse Embryo Recovery
Transgenic Mouse Facility
Fuming Pan, Director
505 S. Goodwin Ave., 15 Morrill Hall, Urbana, IL 61801
Phone: (217) 244-0649 FAX: (217) 244-0466
Email: fpan@uiuc.edu
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