Frequently Asked Questions
Q: Where is the Transgenic Mouse Facility located?
A: The Transgenic Mouse Facility (TMF) consists of office, lab, and animal holding space in the basement of Biological Sciences III at the main UC-Irvine campus. For a PDF version of the campus map click here. Bio Sci III is 519. Our new shipping address is:
UC-Irvine
ULAR
121 BSA
Irvine, CA 92697-1310
Q: What should I do to have a transgenic mouse made?
A: Please note that we are now performing the final DNA purification step ourselves, so our requirement for DNA submission has changed. Submit the following to Tom Fielder:
- a signed Service Request Form (please request the latest form from Tom Fielder and mail or fax the completed, signed form to him)
- a restriction digest of at least 50 micrograms of your plasmid, with a gel picture showing an aliquot of the digest to demonstrate completion of the reaction, and clearly labeled to indicate which band is the transgene
- documentation showing institutional approval for use of the transgenic animals and for making the recombinant DNA.
- For non-UC clients, an MTA is also required – please request the form from Tom Fielder.
Q: How long does it take to obtain founders from DNA microinjection?
A: This can vary quite a bit, depending on several factors. The minimum turnaround time, from receipt of the DNA to delivery of the first offspring, is about 8-9 weeks (2 weeks to get the egg donors and superovulate them, 3 weeks for pups to be born, and another 3-4 weeks before they can be weaned and shipped). It can be longer, depending on how many orders are scheduled ahead of yours. Also, first priority is given to UCI clients and jobs in progress. For clients who prepare their own DNA, the quality of the DNA construct prep and the accuracy of your concentration estimate can also have a significant impact on turnaround time. In any case, injections cannot be scheduled until a completed Service Request Form, the necessary documentation, and a suitable DNA prep have been received by the TMF.
Q: What should I do to have a knockout mouse made?
A: We offer all the services necessary to introduce a targeting construct into ES cells, select clones, characterize them by Southern blot, expand selected clones for long-term storage, prepare clones for microinjection, and inject ES cells into blastocysts to make chimeric mice. We strongly urge clients to consult with us prior to making targeting constructs. Alternative methods of obtaining targeted ES cells may be preferable in some cases.
ES cell clones for microinjection should be sent on dry ice to Tom Fielder at the above address (they will be stored in liquid nitrogen). The number of cells in each vial should be roughly equivalent to a 60mm plate of good-sized ES cell colonies. Plan to send at least 2 vials per clone. One vial is needed for pathogen testing.
Q: Can you make transgenics for investigators who are not part of the UC system?
A: Yes! The patent on DNA microinjection to make transgenic animals expired on 10/10/06. We can now perform DNA microinjection to make transgenic mice for any outside institution, non-profit or otherwise.
Q: How can I follow the progress of my project?
A: Tom Fielder is the primary contact for all questions regarding the progress of individual services. Shuling Wang can also answers questions about our gene targeting services and the progress of gene targeting jobs. Feel free to contact us at any time. Email is the preferred medium of communication.
Q: How should I prepare my DNA construct?
A: Once you have determined the construct was correctly engineered (e.g., by sequencing, restriction mapping, and/or functional testing), you should do a standard plasmid prep to obtain at least 50 micrograms of clean DNA. Digest 50 micrograms with suitable restriction enzyme(s) to release the transgene from the plasmid backbone. Run a small amount of this digest on an agarose gel to check for completion. If the digest looks good, send the remaining reaction mix and the gel picture to us. We will perform the final purification of the transgene DNA fragment.
Q: How should I test my construct?
A: The usual methods for testing constructs are as follows: sequencing the DNA, doing in vitro expression, injecting it into frog oocytes. None of these methods will guarantee that you will get expression of your transgene in the founders. However, they can reveal mistakes in design or engineering that prevent expression. One method for testing new promoters is to join them with a reporter gene (e.g., lacZ or GFP) and make "transient" transgenics, i.e., check for expression in mouse embryos. This can cut 4-5 weeks from the normal turnaround time.
Q: What are my chances of getting a transgenic founder?
A: Although we cannot guarantee expression of the transgene, the Premium DNA microinjection service does guarantee to produce at least 3 transgenic founders, or a total of 50 pups, whichever comes first.
