2. Run out a few hundred nanograms of DNA on a minigel to determine that the digest went to completion and that the bands are the correct size. Upload a photo of the minigel when you fill out the submission form. Mark the bands on the photo to show which band should be purified for microinjection..
3. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to the Transgenic Core lab and we will purify the DNA for microinjection from the digest.. We use the NucleoSpin kit (Macherey-Nagel) to purify microinjection DNA. Please note, if you want use large DNA fragments such as bacterial artificial chromosomes, that there is a different protocol for the preparation of the BAC DNA for microinjection.
4. Upload a gel photo when you fill out your submission form that shows you have a PCR assay for an endogenous mouse gene such as beta-globin. All DNA samples from potentially transgenic mice should give a positive result with an endogenous gene PCR. This will ensure that no transgenic founders are discarded because PCR inhibitors co-purified during DNA isolation from tail tip biopsies.
5. Upload a gel photo when you fill out your submission form that shows your genotyping PCR can detect the transgene at the 0.1 copy level when it is mixed with mouse tail DNA. Upload your calculations of copy number along with the gel photo. If you need tail tip DNA to set up the assay, we will give you some..
6. Print out your online submission form from the MiCores portal and bring it to the lab when you drop off your restirction enzyme digested transgene.
7. Drop off all your materials at the microinjection lab: Room 2526, Building MSRB I.
Transgene constructs are purified, quantitated, and microinjected into (C57BL/6 X SJL)F2 mouse eggs and surgically transferred to recipients. Other mouse eggs can be used for transgenic production when prior arrangements are made with the Core. Contact Thom Saunders regarding custom mouse strains.
Transgenic mouse production is a fee for service provided by the Transgenic Core. The Core prioritizes all requests for service on a “first-come, first-serve” basis. Your DNA will be added to the microinjection queue in the order that it is received.
Targeting vector DNA are used to modify genes in mouse embryonic stem cells.This process is complex, involving many procedures that are carried out over a year or more. Careful attention to detail in the design stage often makes the difference between a smooth, successful experience and a harrowing, successful experience. Investigators are invited to contact Thom Saunders for tips on planning an experiment.
3. Bring the linearized targeting vector DNA and submission form to the Mouse Embryonic Stem Cell Laboratory (2578 MSRB II).