RRC-TPS: DNA Microinjection
- The investigator must open both an RRC account and a BRL account. The BRL account will be used to purchase and house the required mice. The investigator must also review our User Policies.
- The investigator must submit a UIC Animal Use Protocol for approval by the Animal Care Committee (ACC) before initiation of this project. When preparing your Protocol you should refer to the TPS ACC Protocol for generating transgenic founders (contact TPS Director). Any plans for experimental usage of transgenic mice subsequent to the generation of founders must be described in your Protocol application and approved by the committee.
- The investigator must complete the Application for DNA Microinjection (contact TPS Director). In addition, the investigator must read our protocol for preparing DNA for microinjection, request from the TPS an aliquot of Injection Buffer (in which the DNA must be dissolved), and prepare the DNA fragment for microinjection.
- When the prepared DNA, accompanied by a completed Application form, is submitted to the TPS and the facility has confirmed the concentration, size and integrity of the DNA, the investigator will be informed as to when the microinjections will be performed. Mice will be ordered (using the investigator's BRL account) to arrive 1/2- to 1-week ahead of time. The TPS will take responsibility for keeping an accurate daily account of the number of mice being housed under the investigator's BRL account.
- Embryos will be microinjected with a single DNA construct and transplanted into pseudopregnant females over a 3-day period. Approximately 200 injected embryos are transplanted into 10 foster (surrogate) mothers, although variability with regard to these numbers should be expected (note: occasionally, due to unforseen problems, additional mice may have to be ordered and injections carried out over a longer period of time).
- The investigator will be notified when the microinjections have been completed and when the potential litters are due (19 days after microinjection). If the investigator desires to follow the pups from birth, please let the TPS know at this time so the appropriate arrangements can be made. We encourage investigator participation to identify phenotypes associated with transgene expression in founders.
- The TPS will monitor the pregnancies. The investigator will again be notified when all the recipient mothers have delivered (approximately 60%-70% of recipients carry the microinjected embryos to term; litter size is variable). Tail biopsies will be performed at weaning (~21 days postbirth). When all litters have been weaned the tails will be turned over to the investigator for DNA isolation and transgene analysis. Nontransgenic tails will be included for negative controls if requested. We strongly recommend that you read our comments on transgenic screening and make sure the appropriate positive controls are included in your screening strategy. Note that per diem will be charged during the time it takes the investigator to complete the analysis.
Once the tail DNA analysis is complete the investigator may arrange to meet with a member of the TPS to identify the founders, and the facility will request a transfer of the founders out of the TPS to the care of the investigator. Prior to this time please be sure to make the appropriate arrangements with the BRL for housing your mice. BRL staff will be responsible for transporting the mice. Note, for some housing locations on campus mice must be screened for murine pathogens prior to the transfer. Fees associated with these tests will be charged to the investigator's BRL account.
Under normal circumstances our microinjection schedule should yield an average of 5 transgenic mice. However, several factors can affect the production of transgenic mice, for example, the purity and the concentration of the DNA to be microinjected; the mouse strain used for transgenic production; or the potential for embryonic or neonatal lethality in the case of transgenes expressed during embryogenesis. Please advise us of potential lethal or other phenotypes. If two founders are not produced from the first round of injections, a second round will be performed at the Investigator's request (Production Fee waived, investigator must pay for additional mice and per diem). This is only after we discuss the screening analysis and are convinced that positive transgenics were not missed due to procedural error. The investigator must provide clear evidence that the screening assay will detect the transgene down to the level of one (hemizygous) copy per genome. Please see Director for advice pertaining to the appropriate controls. Potential embryonic lethality will also be considered when deciding on a second round of microinjection. Under some circumstances we may ask the investigator to submit a new sample of DNA for the second round of microinjection. Any further attempts past the second round to produce founders are discouraged and would require additional payment of all charges.
- Please acknowledge the Transgenic Production Service if you use mice produced in our facility in a publication.