Targeting Vector Submission for ES Cells
1. We will purify the DNA for you. Simply perform a restriction enzyme digest on your cloning vector to liberate 50 ug of the transgene insert from the cloning vector.
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 (Clonthech) 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
Core. The Core prioritizes all requests for service on a "first-come,
basis. Your DNA will be added to the microinjection
queue in the order that it is received.
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