Transgenic mouse production by microinjection of oocytes  Gene targeting in ES cells  Generation of ES cell chimeras  Sperm & Embryo cryopreservation / Recovery  Rederivation of mouse strains to achieve pathogen-free status  Other Services Transgenic mouse production by microinjection of oocytes 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. Although a variety of procedures are acceptable for this purpose, including electroelution, glass-milk adsorption, and recovery by agarase treatment, it is recommended to follow the procedures 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 TrisHCl, 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 will inject at least 200 zygotes with the DNA fragment. Genomic DNA will be prepared from tail biopsies of 3-4 week old offspring by proteinase K digestion/phenol extraction/ethanol precipitation. The Core guarantees at least two transgenic founder per injection experiment. If no transgenic founders are obtained, the Core will discuss with the Investigator the further proceeding. 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 routinely uses oocytes from B6D2F1 F1 hybrid females, mated to C57Bl males for injection experiments, yielding transgenic founders on a 75% C57Bl genetic background. By request, the Core also can generate transgenic founders on a C57Bl/6 or FVB background. Fees for generation of C57Bl transgenic founders, for injection of non-standard DNA constructs, such as BACs, or other non-standard procedures may vary. To initiate an experiment, the TG Core Group Leader and submits a completed order form for zygote microinjections. Injections will be performed at the earliest possible time, usually beginning within 4 weeks after receipt of the DNA construct. Offspring will be born 3 weeks after injection, and DNA will be made available, 4 weeks thereafter. Fee information is available from the TG Core Group Leader. BACK TO TOP Gene targeting in ES cells The Core performs gene targeting experiments in D3 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 D3 ES cells with selection in G418 and Gancyclovir, but other cell lines and selection procedures are available upon request. To initiate an experiment, the Investigator contacts the TG Core Group Leader 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 available from the Core Director. 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. BACK TO TOP Generation of ES cell chimeras 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 mycoplasm contamination, and culture conditions must be provided by the Investigator. The Core will prepare the ES cells for injection into C57Bl blastocysts will inject at least 50 blasts per injection experiment. Offspring will be turned over to the Investigator at weaning age (4 weeks). Successful production of chimeras (as determined by visible contribution of ES cells to hair coat) is only guaranteed if the transgenic ES cells have been generated by the Core. If no offspring is obtained, the experiment is repeated at no cost to the Investigator. To initiate an experiment, the Investigator contacts the Core Director 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 coat color may be assessed 3 weeks thereafter. Fee information is available from the TG Core Group Leader. 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. BACK TO TOP Sperm & Embryo cryopreservation / Recovery The core has the expertise and equipment to cryopreserve fertilized oocytes and sperms. To plan and arrange for preservation experiments, contact the TG Core group leader. BACK TO TOP Rederivation of mouse strains to achieve pathogen-free status The Core can assist in rederivation experiments by performing blastocysts transfers, or Caesarian section. Rederivation must be coordinated with ARC staff. To plan and arrange for preservation experiments, contact the TG Core group leader, and the ARC. 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 Investigatorís needs. To maximize chances of successful rederivation, the stud males must be older than 2 months of age, and less than a year old. Males should be housed individually for two weeks 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 mating for rederivation. Superovulation works best on sexually immature females in a narrow window at 3 weeks of age, and on sexually mature young females. Thus, females should be either 3 weeks old or 6 to 10 weeks old. Rederivation includes the breeding of males with superovulated females, collection of pre-implantation stage embryos, washing/culturing of embryos, 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 (4-6 weeks after mating). BACK TO TOP Other Services The core has the expertise to perform IVF. IVF can be used to rapidly expand mouse lines in strains that may be poor breeders or when few breeders are available. Please contact the TG Core group leader to make arrangement. BACK TO TOP