Transgenic Core Facility
The Transgenic Core consists of a laboratory for procedures involving production and maintenance of transgenic mouse strains, a tissue culture facility for all procedures involving Embryonic Stem (ES) cells and gene targeting experiments, and a transgenic barrier holding facility.
The Core is under the direction of Dr. Weiler, who has extensive experience in the production of genetically altered mice, and includes three additional staff skilled in various aspects of transgenic animal production and related procedures.
The laboratory is located in the vivarium of the Translational and Biomedical Research Center at MCW and is equipped to perform all procedures involving production and maintenance of transgenic or knock-out mice. Instrumentation includes 3 injection workstations (Nikon inverted phase contrast microscope with Hoffman modulation optics, temperature-controlled injection stage, air-suspended injection table, Eppendorf motor-driven injection system), microforge, needle puller, Nikon stereo zoom microscope, surgical microscope, CO2-incubator, laminar flow hood for surgical procedures, and a cell fusion instrument for tetraploid embryo production.
Additional workspace equipped for cryopreservation and gene targeting is located at the Versiti Blood Research Institute.
Liquid nitrogen tanks and an automated cell freezer for cryopreservation of fertilized oocytes are available as shared instrumentation through the Transgenic Core. Over the past three years, the Core has successfully performed 24 different gene targeting experiments, produced more than 14 germline-competent ES cell aggregation chimeras and gene targeted mouse strains, generated transgenic animals via zygote micro-injection, and assisted in routine animal procedures such as re-derivation of mouse strains. All services (including gene targeting, blastocysts injections, and zygote microinjections) are available to MCW-associated Investigators on a fee-for-service basis.
A number of very useful protocols for the generation and analysis of transgenic animals are described in Manipulating the Mouse Embryo: a laboratory manual by B. Hogan et al., 2nd edition or newer, published by Cold Spring Harbor Laboratory press (ISBN 0-87969-384-3).
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 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.
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 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.
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
If you have any questions or need assistance with a technique, please email the Core Supervisor.
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.
Transgenic Core Facility Ordering Information
- Approved Animal Protocol number for transgenic / knock-out mice, including available housing space
- Completed Order Form (this link jumps down to the order forms)
- Service Agreement Form (for On-Campus Investigators). A separate Service Agreement form will be emailed to off-campus Investigators.
- Completed "Condition of Use" form if using gene targeting/blastocyst injection service
- Documentation about experimental materials provided to the Core
- Documentation about established screening procedure to characterize transgenic offspring or ES cells.
The project will then be assigned a project ID, and placed in the work queue. Upon initiation of the experiment, you will receive regular updates about the project progress. At project completion, the Investigator will receive a summary and a copy of our laboratory notes.
The following forms may be filled out online and emailed directly to the Core Supervisor. If you are having problems emailing them, please print them out and fax them to the number listed below. If you have any questions or concerns, please contact the TC Core Supervisor.
- Blank Project Request (PDF)
- Blastocyst Injection Request (PDF)
- Conditions of Use Statement (PDF)
- E14Tg2a.4 ES Cells Experiment (PDF)
- Gene Targeting Request (PDF)
- Mouse Line Rederivation Request(PDF)
- Sperm Cryopreservation Request (PDF)
- Zinc Finger Injection Request (PDF)
- Zygote Microinjection Request (PDF)
The Core is not a business, and does not attempt to generate net revenue. The Core’s only mission is to support and enable experiments by other Investigators. Fees are levied to cover approximately 50% of the actual cost involved in performing these experiments. The production of transgenic or knock-out animals is a long-term, multistep process, and in most cases the cost of generating the mice is only a fraction of the total project expense. We strongly recommend taking these considerations into account when applying for funding. The fee for individual services depends on the scope of the project. For details, please contact the Core Director.