Transgene Sample Submission Instructions
Targeting Vector Submission for ES Cells

Home

Transgenes

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.

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. Bring a photo of the minigel to submit with your sample. 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 Core lab and we will purify the DNA for microinjection from the digest. We use either the Gel Extraction Kit (Qiagen) or the Machery Nagel Nucleospin extract kit (Clontech) for purification of 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. Bring a gel photo 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 make sure that no transgenic founders are discarded because PCR inhibitors co-purified with the genomic DNA.

5. Bring a gel photo that shows your genotyping PCR. Test the PCR against 10, 1, 0.1 and 0.01 copy standards. The assay must  detect the transgene at the single copy level, preferably 0.1 copies, when it is mixed with mouse tail DNA. Bring  your calculations of copy number along with the gel photo. If you need tail DNA to set up the assay, we will give you some.

6. Bring a submission form with your contact information and billing information.

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 Vectors

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.

1. Prepare the targeting vectorDNA as described..

2 Fill out the electroporation submission form.

3. Bring the linearized targeting vector DNA, the submission form, and backup materials (evidence of a screen to detect homologous recombination in 96-well plate ES cell DNA) to the Mouse Embryonic Stem Cell Laboratory (2578 MSRB II).

 
Top Home

Transgenic Outline | Gene Targeting Outline |Laboratory Protocols | List of Services
Pertinent Publications | Service Description | Genotyping Mice|Mouse Breeding Suggestions | Links | Home  | UMMC Home Page


comments to: tsaunder@umich.edu