UCI > Research > TMF > FAQ
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 the Gillespie Neuroscience Research Facility in the College of Medicine at the main UC-Irvine campus. For a PDF version of the campus map click here. The building number for Gillespie is 837.
Q: What should I do to have a transgenic mouse made?
A: Submit the following to Tom Fielder:
- a signed Service Request Form (fill this out on-line here, print, sign, and mail or fax to Tom Fielder)
- your DNA construct (at least 1 microgram), with a gel photograph showing its purity and concentration (as compared with a suitable DNA mass ladder)
- documentation showing institutional approval for use of the transgenic animals and for making the recombinant DNA.
Q: How long does it take?
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 much longer, depending on how many orders are scheduled ahead of yours. Also, first priority is given to UCI clients and jobs in progress. 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 now 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 3 vials per clone.
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. Julie Underwood may also be able to answer questions about genotyping and shipping.
Q: How should I prepare my DNA construct?
A: Once you have determined the construct was correctly engineered (e.g., by sequencing or restriction mapping), you should do a standard plasmid prep, cut the construct out of the plasmid backbone, separate the two pieces electrophoretically, and purify the linearized construct by any of the standard techniques, e.g., Qiagen, GeneClean, phenol/chloroform extraction and EtOH precipitation, sucrose gradient, or cesium chloride gradient. The success of the project does not seem to be heavily dependent on which technique is used, but rather how carefully it is done. The final step should consist of filtration through a 0.45 or 0.2 micron filter. I recommend 0.45 or 0.2 micron Millipore ultrafee-MC filters (catalog numbers are UFC30HV00 for 0.45, UFC30GV00 for 0.2, packs of 100 filters). It is critical that you do NOT use filters that have been sterilized with ethylene oxide; therefore I recommend using unsterilized filters. The concentration must then be determined, in most cases, by running several amounts of the construct along with a DNA mass ladder (e.g., Invitrogen catalog #10496-016), and comparing the staining intensities. Most preparations will not be concentrated enough to read accurately on a spectrophotometer. It is preferable to send the DNA at a concentration of at least 20 ng/microliter. The final dilution will be made by the TMF prior to injection. Errors in concentration of as little as 2-fold can severly diminish the chances of producing a transgenic founder.
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. A similar guarantee is offered for ES cell injection. Out of more than 2200 pups produced to date via DNA microinjection by the TMF, 20% have been transgenic.
