Pronuclear Injection Services
- The investigator will provide 5 µg of transgenic plasmid digested with appropriate enzymes to release the transgenic fragment from the vector backbone as an ethanol precipitate. BACs are submitted as purified supercoiled DNA. Purity and integrity of the DNA will largely influence the success of the project. To this end, documentation of purity will be required upon submission of your sample. Agarose gel analysis is sufficient for cDNA projects but does not provide integrity information for BACs. Therefore a pulsed field gel analysis of both un-cut sample and sample digested with a rare cutter (such as NotI) is required for BAC transgenics. Alternatively, the Transgenic and Gene Targeting Core can perform purity testing for a service fee.
- The Core will generate 3 transgenic founders or 50 non-transgenic pups, whichever is achieved first. In the event that no transgenic founders are produced the project will be re-evaluated.
- A small amount of tail tissue from pups resulting from injections will be provided to investigators for genotyping. It is recommended that investigators perform spiking studies to verify that the screening strategy is capable of detecting a single copy of the transgene in a genomic background to avoid false negatives.
ESC/Gene Targeting Services
- If requesting ESC targeting, the investigator will provide 100 µg of linear DNA for ESC transfection as an ethanol precipitate. The vector should contain arms of homology that are co-isogenic with the background strain of the targeted ESC line. The Transgenic and Gene Targeting Core routinely works with a variety of B6 and 129 substrains. Other lines may be available on request.
- DNA from isolated clones will be provided to the investigator for screening. Both PCR and Southern blot analysis can be used for screening for targeted clones. Southern blots provide quantitative results and can be useful for eliminating targeted clones that also harbor random integrations.
- The Transgenic and Gene Targeting Core will inject up to 100 C57BL/6J or albino C57BL/6J blastocysts per clone.
- It is recommended that investigators check with consortia databases (IMPC, KOMP/EUCOMM) in the event that the desired target is available. In this case, the clones can be sent directly to the Core for expansion and injection. Molecular validation of KOMP/EUCOMM clones is highly recommended. We also recommend investigators order 3 clones for microinjection.
- The Core CANNOT guarantee germline transmission of founders since factors beyond our control can affect transmission. This applies to in-house targeted clones as well as consortia material.
Cryopreservation: Investigators who wish to decrease mouse census and preserve valuable strains can utilize the Core’s cryopreservation services.
- Embryo cryopreservation can be achieved using either natural mating-derived embryos or embryos derived from in vitro fertilization (IVF). Embryo cryopreservation is accomplished in the Core by vitrification of 8-cell embryos, a process that bypasses the formation of damaging ice crystals during cooling. Consultation with Core staff is recommended in order to determine which protocol will be most appropriate for specific background strains.
- Alternate tissues can also be cryopreserved such as sperm and ovaries.
In many cases, the Transgenic and Gene Targeting Core can accommodate special requests and project needs. Please refer to the Fee Schedule for a complete list of available services and à la carte fees. Special requests for additional services will be handled on a consultation basis. Please contact Dr. Kerri Kluetzman for further information. Letters of support can be provided to investigators preparing to submit grant proposals to funding agencies.