Knockout Mice

The generation of gene-knockout mice, wherein one or more of their genes have been made inoperable, is another method to study gene function. Gene targeting is carried out in mouse embryonic stem cells (ES cells) that have been genetically altered in vitro through custom-designed DNA constructs. Some types of constructs can be made to allow for tissue-specific expression.

These modified ES cells are then injected into mouse blastocysts, surgically placed into the oviducts of recipient female mice, and allowed to develop to term. Tissue-specific gene modification is also often possible. Mouse pups carrying genetic material from the contributed ES cells have a unique coat color pattern. These are subsequently transferred to the investigator for breeding and analysis.

Production of Knockout Mice

Option A: For labs without ES cell culture experience

1. The investigator makes the construct (contact the transgenic facility to obtain a high efficiency targeting vector), purifies the DNA by phenol: chloroform extraction, and figures out screening PCR conditions (1 fg sensitivity). The investigator is required to provide the facility a construct map and a gel photo with purified DNA plus PCR bands to document purity of the DNA preparation and readiness of the PCR screening conditions.
2. The facility performs ES cells transfection and culture.
3. The facility will then pick transformed clones, with a maximum of 500 clones in cases of low efficiency.
4. DNA lysates from the clones will be given to the investigator for screening by PCR, which must be done within 24 hours. If PCR does not give conclusive results, the facility will pick extra colonies onto 96-well plates for screening by mini-Southerns. Investigators are expected to do this procedure, but the transgenic staff can do the Southerns for an additional charge. Results are required in 2 weeks.
5. Once positive clones are identified by PCR or mini-Southern, the facility is notified and the clones will be expanded for DNA analysis by Southern blotting.
6. The investigator is required to perform genomic Southern blot to confirm the knock-out configuration before blastocyst injection.
7. The facility performs blastocyst injection of up to 5 clones, with 16 blastocysts/clone, to facilitate obtaining the germline chimera from at least 2 independent clones.
8. High degree chimeric mice will be transferred to the investigator 3 weeks after birth for further breeding and testing.

Option B: For labs with ES cell culture experience

The investigator will perform the first 6 steps, and the facility will then start from step 7. In this case, because the ES cells are cultured outside the Transgenic facility, there are fewer guarantees regarding the outcome. The service charges for the applicable time and materials provided by the Transgenic facility still apply.

Option C: Cre/Flp/FC31 excision P

1. The facility will electroporate cells with the Cre/Flp/FC31 expression cassette and pick 24 clones for further testing.
2. The clones will be tested by the investigator by PCR within 24 hours and genomic Southern blot for correct configuration. The Southern blot results are required within 2 weeks.
3. The facility will freeze 3 clones while the tests are going on. After confirmation of the desired genetic modification, the facility will perform blastocyst injection. As above, chimeric mice will be transferred back to the investigator 3 weeks after birth.

Note: Animal costs and per diem care of mice used in meeting services from the Transgenic facility will be billed to investigators according to AHR's standard rates.