L.T. Christian III Transgenic Mouse Core Facility
Jolene Windle, Ph.D.
Resource director
(804) 828-5843
The primary purpose of the Transgenic/Knock-out Mouse Shared Resource is to provide an efficient and economical means for producing genetically modified mice for members of the Massey Cancer Center. Genetically modified mice (including transgenic mice, in which customized genes are introduced into the genome, or “knock-out/knock-in” mice, in which endogenous genes within the mouse genome are specifically inactivated or modified) have become an indispensable tool in cancer research. The utility of such models ranges from studies of the basic biology of tumorigenesis and progression to the creation of genetically accurate tumor models for the evaluation of novel therapeutic approaches.
Facilities and equipment
The Transgenic/Knock-out Mouse Shared Resource occupies approximately 1,000 square feet of dedicated, controlled access space on the 8th floor of the Medical Sciences Building. The core includes the following rooms:
- A 225-square-foot microinjection lab, containing a Nikon Diaphot microscope with DIC optics, a pair of Narishige micromanipulators, a Kinetic Systems air-table, a high resolution black and white video camera and monitor, a Nikon SMZ-800 stereo microscope with teaching head (for mouse surgery), a Nikon SMZ-645 stereo microscope (for embryo manipulation), a Sutter needle puller, a Narishige microforge, a Narishige beveller, a Forma CO2 incubator and a fume hood.
- A 225-square-foot tissue culture lab, containing a Coulter Z1 particle counter, a BioRad gene pulser II electroporator, a Nikon TS100 microscope, a Nuaire tissue culture hood, a Forma CO2 incubator, a tabletop centrifuge and a refrigerator/freezer.
- A 120-square-foot procedure room containing a fume hood (for ear notching and tail clipping).
- A 145-square foot animal holding room (dedicated to core mice only).
- A 150-square-foot clean cage holding room.
- A 75-square-foot office for the research associate, containing a fully networked Dell computer.
The molecular biology work associated with mouse production (DNA fragment purification, tail DNA isolation, Southern blot and PCR analysis) is carried out in Dr. Windle’s research lab, which is on the floor immediately below the animal space in the Medical Sciences Building. Her lab consists of approximately 900 square feet of laboratory space and is fully equipped for molecular biological studies.
Services provided
The Transgenic/Knock-out Mouse Shared Resource offers the following services:
- Transgenic mouse production: The core generates at least three founder mice for each transgene.
- Knock-out/knock-in mouse production: The core can perform all or a subset of the following procedures: ES cell electroporation, ES cell culture under appropriate selection conditions, expansion of drug-resistant clones, PCR/Southern blot screening of clones for the correct recombination event, re-electroporation with a cre expression vector to delete floxed selection markers and subsequent re-screening of clones, injection of targeted ES cell clones into blastocysts for chimeric mouse production. For projects in which the core has performed the ES cell targeting, at least two independent ES cell clones are generally injected into blastocysts, and a minimum of five chimeras are generated.
- Mouse line rederivation: Lines of pathogen-infected mice are rederived by embryo transfer to generate specific pathogen-free mice.
- Sperm cryopreservation: Lines of mice are cryopreserved by collection, freezing and long-term liquid nitrogen storage of sperm. When needed, re-establishment of the line can be accomplished by thawing sperm and performing in vitro fertilization of eggs isolated from WT females.
- Tail DNA genotyping: The core isolates genomic DNA from tail biopsies (or other tissue) provided by the investigator and performs PCR genotyping.
- Consulting services: The core provides consultation on many aspects of transgenic/knock-out mouse technology, including experimental design, vector design, budgeting for mouse colony maintenance and preparation of applications to the IACUC for the use of laboratory animals in research. In addition, the core staff provides instruction on overall colony management, including breeding strategies, weaning, ear punching and tail DNA isolation.
Charge-back schedule
- Transgenic Mouse production: Investigators are charged $3,000 per construct for the production of transgenic mice. An additional $500 is charged for production of transgenic mice in a C57Bl/6 or other inbred background.
- Knock-out/knock-in mouse production: The cost for the three stages of knock-out/knock-in mouse production is:
- Stage 1 - ES cell electroporation: $3,000 per targeting vector construct; $1,500 for cre expression vector re-electroporation.
- Stage 2 - screening of ES cell clones: $2,000 for three 96-well plates; $1,000 for screening after cre expression vector re-electroporation.
- Stage 3 - blastocyst injection: $3,000 for two clones.
- Mouse line rederivation: The charge for rederivation of a line of mice by embryo transfer is $1,000.
- Sperm cryopreservation: The charge for cryopreservation of sperm from two males of a single line and long-term storage is $750.
- Tail DNA genotyping: The core charges $5 per sample for genomic DNA purification and an additional $5 per sample for PCR genotyping.
- Consulting/support services: There is no charge for these services.
