General Information

The UNC Animal Models Core Facility was established to provide mouse genetic modification services, reagents and related technologies. Services for creating transgenic mice include isolation of the transgene fragment and pronuclear microinjections. Animals are typically a C57/Bl6 X C3H hybrid strain, but can be made in a specific strain if needed for an additional cost, which varies depending on the cost of the strain. Gene targeting services include assistance with the design of targeting vectors, creating gene-targeted ES cells and blastocyst microinjections of the ES cells to create tarnsmitting chimeras. Related services include creating homozygous ES cells from targeted ES cells, Cre- or Flp-mediated excision in ES cells, karyotyping, tail DNA purification, PCR screening, Southern blotting, embryo/ovary/sperm cryopreservation, ovary transplants and in vitro fertilization. Reagents available include mouse genomic DNA (for PCR and Southern hybridization assays), a mouse genomic library (strain 129/SvEv in l phage) and several plasmids useful for engineering transgene and gene targeting vectors. To obtain transgenic mouse services, clients must fill out a "Transgenic Mouse Request and Agreement"  and a "Transgene Assay" form.  The first form assures the facility that an "Application to Use Live Vertebrate Animals" covering the genetically modified mice to be made by the facility has been approved by the Institutional Animal Care and Use Committee (IACUC). It also identifies the grant or source of funds to pay for reagents and services, a contact person in the client's lab and acts as a contract between the facility and client. The second form provides the facility with documentation that the client is able to detect the transgene in the background of genomic DNA. Copies of the forms and a price list are attached.  A description of the specific services available, the responsibilities for the client, the reagents needed from the client and limitations of the services is attached, as is a list of protocols available from the facility.

Transgenic Mouse Services

            The UNC Animal Models Core Facility creates transgenic mice from DNA provided by the client.

1. Submission of DNA

     For an optimum number of founders, the transgene plasmid should be purified using an endotoxin-free system, such as the Qiagen EndoFree system (catalog number 12362). All or most of the plasmid backbone should be cut from the transgene DNA with restriction enzymes in such a way that the transgene DNA is easily separated from the plasmid backbone by agarose gel electrophoresis. Clients can either provide the facility with the gel-purified transgene in sterile injection buffer (10 mM Tris/ 0.1 mM EDTA, pH 7.5), or can supply the facility with the cut DNA. In the former case, the client must supply a minimum of 500 ng of DNA at a concentration of not less than 10 ng/ml. In the latter case, the facility will isolate the transgene fragment. If the client elects to have the facility isolate the transgene (preferred), they should supply a minimum of 20 micrograms of cut DNA to the facility and must supply the facility with an ethidium bromide-stained agarose gel picture showing the digestion pattern  as well as a rough map of the plasmid. All transgene DNA's will be tested for toxicity before injections are performed. Those that result in high levels of embryo lysis will be re-isolated. If transgene-based toxicity is suspected, the DNA may not be injected.

2. Pronuclear Microinjections

     The transgene DNA will be injected into the pronuclei of C57/Bl6 X C3H hybrid embryos and implanted into pseudo-pregnant recipient females. Strains other than the hybrid strain can be used, but will incur additional charges for the costs of purchasing (including shipping) and breeding the animals, if necessary. All clients are required to complete a "Transgenic Mouse Request and Agreement" form and a "Transgene Assay" form before animal work can be initiated (see attached copies). The client's approved Application to Use Live Vertebrate Animals number (IACUC number) will be placed on all cages containing animals belonging to the client. This means that the client will begin to accrue charges from the Division of Laboratory Animals (DLAM) as soon as animal work for the client has been initiated. The client is responsible for payment of all DLAM charges. Pups are born approximately 3 weeks after the injections and tail or toe biopsies are provided to the client at approximately 10 days after the birth of the pups. Clients are required to screen the pups to identify potential founders. For this reason, the client must demonstrate a reliable genotyping assay (by PCR or Southern hybridization) before pronuclear injections commence. Results of the genotyping screen must be reported to the facility within 10 days after receiving the biopsies, or prior to weaning of the pups (21 days of age). The goal of the facility is to generate a minimum of 4 transgenic animals for each experiment. This is necessary for a successful experiment because 25-30% of transgenic animals typically do not express the transgene and because of variability in transgene expression levels in the founders. If no transgenic animals are obtained from 400 injected embryos, a consultation with the facility director is advised. At approximately 3 weeks of age (weaning) all pups will be transfered into the clients care, whether or not the genotyping is completed, unless other arrangements have been made with the facility. After this time, colony management fees will be assessed to the client until all animals are transferred into the care of the client. Because the number of pups generated can be in excess of 100 and the colony management fees can be quite high, clients are encouraged to genotype the animals quickly. Animal space at UNC must be requested from DLAM and is currently assigned by Dr. Dwight Bellinger ((966-3111). Be advised that animal room space cannot be assigned by animal room caretakers. The facility will work closely with the client if unusual circumstances prevent the procurement of animal space. However, failure to secure animal space in a timely manner will result in additional charges and could result in the loss of valuable animals.