The Investigator provides purified DNA suitable for microinjection experiments, appropriate documentation about fragment preparation, and documentation of an established protocol for identifying transgenic founder animals carrying the transgene.

If at all possible, the DNA fragment to be injected must be separated from plasmid -vector components by restriction digest, followed by agarose gel electrophoresis and recovery of the fragment from the gel. There are a variety of procedures that are acceptable for this purpose, including the QIAEX II™ gel extraction kit or Mo Bio UltraClean™ GelSpin® Kit, electroelution, glass-milk adsorption, and recovery by agarase treatment, which are described in Manipulating the mouse embryo: Hogan, B. et al.; Cold Spring Harbor Laboratory Press. 2nd edition, pages 228-232 (see protocols). Only reagents of highest available quality should be used. The final elution of the fragment should be performed with injection buffer [10 mM Tris-HCl, pH 7.4; 0.1 mM EDTA] prepared with embryo-certified water (SIGMA). The Investigator provides the purified fragment in injection buffer at a concentration of > 30 micrograms/ml . The amount and purity of the purified fragment is determined by UV absorption (spectrum from 220 to 300 nm), and by comparative gel analysis. The latter is achieved by running an aliquot of the fragment on an agarose gel side by side with various known amounts of the parental plasmid digested to release the fragment to be analyzed. The concentration of the isolated fragment can be judged by comparing the relative intensities of the bands after ethidium bromide staining. Documentation about fragment preparation and analysis (functional vector map, size, relevant restriction sites, UV spectrum, Gel analysis) is submitted to the core with the fragment.

Documentation of a genotyping protocol for identifying transgenic founder mice by southern blot analysis or by PCR-analysis of genomic DNA must be provided by the investigator before initiation of injection experiments. Functional assays for detection of the transgene are not sufficient, and not required.

The Core guarantees to inject 200-300 embryos per injection experiment. The lab can request that the Core collect tail biopsies of 3-4 week old pups. We will then extract genomic DNA from the tissue sample by proteinase K digestion/phenol extraction/ethanol precipitation and hand the DNA to the lab for screening. If no transgenic founders are obtained, the Core will discuss with the Investigator how to further proceed. We do not guarantee expression of the transgene. A founder is defined as any mouse with either the full or partial DNA construct.

The Core performs gene targeting experiments in E14Tg2.a and JM8.N4 ES cells. The Investigator provides 100 micrograms of linearized targeting construct and documentation about targeting vector design, preparation of linearized DNA, and selection markers used in the targeting construct. The targeting construct will be transfected into ES cells provided by the Core. Approximately 4 weeks after transfection, the Investigator will receive genomic DNA samples from not less than 250 selected ES cell clones. Screening for targeted ES cell clones will be performed by the Investigator. If compatible with the experimental design, controls for efficacy of the selecting process will be provided by the Core. The Core will retain frozen ES cell stocks until completion of the screening process. The Core commits to providing the above number of selected ES cells, but cannot guarantee successful gene targeting.

The Core performs gene targeting experiments routinely in germline-competent E14Tg2.a and JM8.N4 ES cells with selection in G418 and Gancyclovir, but other cell lines and selection procedures are available upon request.

For Investigators using the E14Tg2a.4 cell line for gene targeting and/or blastocyst injection, you must agree to and sign the "Conditions of Use" agreement from the Mutant Mouse Regional Resource Centers (MMRRC) which can be obtained from the Core Supervisor. This agreement includes acknowledgement of BayGenomics, which donated the E14Tg2a.4 cell line to the MMRRC for distribution, and the MMRRC in any presentations and publications reporting use of the cell line.

For Investigators using the JM8.N4 cell line for gene targeting and/or blastocyst injection, you must agree to acknowledge UC Davis in any resulting publications.

To initiate an experiment, the Investigator contacts the TG Core Supervisor and submits a completed order form for gene targeting. Experiments will be performed at the earliest possible time, usually beginning within 4 weeks after receipt of the DNA construct. DNA for screening will be made available approximately 4 weeks thereafter. Fee information is also available from the Core Supervisor.

Additional services for gene targeting experiments such as expansion of ES cell clones, analysis of modal chromosome numbers, derivation of homozygous recombinant ES cells by selection in high concentrations of G418, and preparation of feeder-cell free ES cells can be provided at the request of, and in collaboration with, the Investigator.

The Investigator provides two vials of frozen ES cells suitable for seeding on 1 well of a standard 6-well cluster plate. If the ES cells have not been generated by the Core, documentation about the absence of mycoplasma contamination, karyotype information and culture conditions must be provided by the Investigator. The Core will prepare the ES cells for injection into C57Bl/6N blastocysts and will inject at least 50 blasts per injection experiment. Offspring will be turned over to the Investigator at weaning age (3-4 weeks). Successful production of chimeras is only guaranteed if the ES cells have been generated by the Core. If no offspring are obtained, the experiment is repeated at no cost to the Investigator.

To initiate an experiment, the Investigator contacts the TG Core Supervisor and submits a completed order form for blastocysts injections. Injections will be performed at the earliest possible time, usually beginning within 4 weeks after receipt of the ES cells. Offspring will be born 3 weeks after injection and tail snips may be obtained 3 weeks thereafter for screening. Fee information is available from the TG Core Supervisor.

Other procedures can on special occasions be arranged in collaboration with the Investigator: this includes embryo aggregation experiments, embryo-ES cell aggregation experiments, and generation of tetraploid embryo-ES cell aggregation chimeras.

The core has the expertise and equipment to cryopreserve sperm. To plan and arrange for cryopreservation experiments, contact the TG Core Supervisor.

The Core uses the JAX® Sperm Cryo Kit for cryopreservation. For best recovery results with sperm cryopreservation, the Core would require two males that are between the ages of 10-16 weeks. They should be proven breeders that have mated and produced pups recently. The males must have been separated from a female for one week prior to the cryopreservation procedure. Older males or males that have not mated can have lower sperm counts or immotile sperm which can reduce the chances of strain recovery when used for IVF. Jackson Labs will perform a quality control test to ensure recoverability of your strain. We strongly recommend not reducing colony numbers until you have received the results of the QC. The sperm will be stored in 2 separate Dewars on the MCW campus as well as at Jackson Laboratory. After 3 years of cryopreservation, a small fee will be charged for storage.

The recovery of sperm from cryopreservation requires an In Vitro Fertilization procedure. This procedure is typically done using wildtype females as egg donors but we can use animals from an Investigator's personal colony, if desired. Mouse strains can vary in their sensitivity to superovulation which can result in low egg numbers when using a strain other than one that the Core is familiar with.

The Core can assist in rederivation experiments by performing embryo transfer surgeries, or caesarian section. Rederivation must be coordinated with Vet staff. To plan and arrange for preservation experiments, contact the TG Core Supervisor, and the BRC vet staff.

Rederivation is used to establish Specific Pathogen Free (SPF) mice. We transfer preimplantation embryos into SPF pseudopregnant recipient females. This procedure is more reliable than caesarian delivery for removal of pathogens. All strains of mice can be rederived, but the efficiency of the procedure depends on the robustness and fecundity of the strain. Inbred strains require larger starting numbers of mice for a reasonable chance of success. The number of mice/breeding pairs required depends on the Investigator's needs. To maximize chances of successful rederivation, the stud males must be proven breeders that are older than 2 months of age, and less than a year old. Males should be housed individually for at least one week prior to mating. If it has been some time since they have mated or if it is their first time, a female should be placed with the male 2 weeks before mating for rederivation, and then removed one week before use for rederivation. Superovulation works best on sexually immature females in a narrow window of 3-4 weeks of age, and on sexually mature young females. Thus, for best results females should be either 3-4 weeks old or 6-8 weeks old. Rederivation includes the breeding of males with superovulated females, collection of pre-implantation stage embryos, washing/culturing of embryos, and the transfer of embryos into recipient females. The Core will provide the hormones for superovulation, perform the hormone injections and mating of the mice to be rederived, provide pseudopregnant recipient females, transfer the embryos to recipients, and house the resultant mice until weaning (3-4 weeks after mating). The recipient females will be housed in quarantine until serology tests can be performed to ensure the SPF status. Serology testing is performed upon weaning of the pups, at 3 weeks of age. The cost of the serology testing is paid for by the requesting Investigator.

The Core Staff has assisted labs with various tasks including:

• General husbandry of a mouse colony
• Mouse tail cutting
• Sub-Q and IP injections
• ES cell targeting and culture
• Embryo vitrification
• IVF

If you have any questions or need assistance with a technique, please email the Core Supervisor.

The Core maintains colonies of C57Bl/6N Stud males for oocyte and blastocyst production; vasectomized males, foster females, neoR-mice for production of neomycin-resistant feeder cells, as well as wild type mice of several defined genetic backgrounds.

A number of different germline-competent ES cell lines are available as frozen stocks.

Frozen stocks of neomycin-resistant primary embryonic fibroblast feeder cells for ES culture.

The core can provide control DNA from ES cells and mouse tails that may be used to establish a screening procedure capable of detecting single copy genes.

The Core can arrange training sessions for individuals to acquire hands-on skills and expertise in the production of transgenic mice, embryonic stem cell culture, and production of ES cell chimeras. The training will enable individuals to successfully conduct these tasks on their own. Specialized training equipment is available.

Training for oocyte and blastocyst injections requires approximately 60 hours over 5-6 weeks. Topics include mouse husbandry, superovulation, harvesting of blastocysts and fertilized eggs, microinjection/blastocyst injection, and transfer of injected eggs or blastocysts into pseudopregnant foster mice.

The ES cell course teaches all the necessary skills to successfully conduct a gene targeting experiment. Topics include preparation of mouse embryonic fibroblast feeder cells, routine culture of ES cells, evaluation of ES cell morphology and ploidy, electroporation, selection and picking of ES cell clones, freezing and DNA preparation. The time frame for this hands-on course is about 5-6 weeks and includes an entire targeting experiment.

We encourage laboratories planning to utilize these techniques on a frequent basis to take advantage of this opportunity. A fee is levied to cover materials, mice, and staff time. Please contact the Core Director to arrange a training session.

The Core has specialized equipment that can upon request be made available to Investigators. This includes a microinjection station, equipment for tetraploid embryo aggregation, a laminar flow workstation with a surgical microscope located in the transgenic animal barrier facility, teaching stereoscope for microsurgery, dissecting microscope with color video capture, fully automated needle puller, a microforge for production of customized needles/pipettes. Access to this equipment depends on utilization by the Core and must be pre-arranged with the staff.