Guidelines for Gene Targeting and Production of Chimeric Mice

Gene KO Information

The Mouse Genome Engineering core facility asks that all investigators follow the instructions listed below when initiating a new project.

1. Please provide us with a brief outline of the intended experiment that describes the DNA construct to be injected and the objective of the experiment. This outline should include a restriction map of your vector and construct.
2. We request that the investigator must make certain, prior to initiating the ES cell targeting work, that she/he has a DNA screening procedure that will detect a region within the recombinant DNA insert and a region outside the DNA insert. We request this must be demonstrated prior to acceptance and placing the construct into the queue.
3. We have Institutional Animal Care and Use Committee (IACUC) approval for the necessary manipulations performed by our staff to generate transgenic animals.
4. You will need to obtain IACUC approval and a protocol number for your intended experiments using your knockout mice prior to our scheduling your project.
5. You will also need to obtain IBC approval for your gene construct. That also must be provided to MGE prior to scheduling your project.
6. The source of DNA should be isogenic to the stem cell line as this results in a higher targeting efficiencies in ES cells.
7. Please, provide the MGEF with a high quality DNA preparation of your targeting construct, restriction digested to separate all vector sequences. Please provide MGEF with 100 μg of the DNA construct well labeled with the construct name, concentration and volume and a gel photograph of the DNA. If you have questions regarding the best procedures to produce the targeting vector or DNA product for electroporation, we can provide you with protocols.
8. We require a cost/center number in order to transfer funds into the MGEF account once we have completed the cell targeting, selection and expansion processes. The charge is $4,000 for electroporation.
9. Once ES cell colonies have been picked and expanded, you will be notified to pick the clones up for screening. During screening a “Master set” of colonies are cryopreserved. Since, these conditions are not optimal for long-term storage of cells, it is important to complete the screening in a timely manner.
10. Once positive clones are identified, notify us immediately and we will give you a second set of cells to re-confirm positive clones. Once re-confirmation is complete, we will expand the clones and place you into the queue of the microinjection schedule.
11. We will notify you when we inject the cells into blastocysts and when pups are born. Once chimeras have been identified, you will be notified when you should expect to have them transferred.
12. Once ES cells have been injected into blastocysts, injections will then be charged to you. The charge is $4,000.
13. Prior to microinjections, make arrangements with Comparative Medicine to have space ready for your mice to be transferred. Use the web based RSS Comparative Medicine order form to order a transfer of the mice to your IACUC # and Cost/center and to have the mice transferred to your mouse room. Should you have questions as to how to do this, contact MGECF or Comp Med.

Should you have any further questions please contact C.B. Gurumurthy. We have protocols that we will provide to you upon request. We have many years of experience with breeding and maintaining genetically altered mouse strains and enjoy sharing our knowledge with you.