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Transgenic/Knock-out Mouse Shared Resource

Mission

Genetically engineered mouse models (GEMMs) have become indispensable tools in nearly all fields of biomedical research.  For example, in cancer research their utility 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. The objectives of the L.T. Christian III Transgenic/Knockout Mouse Shared Resource are to:

  1. Facilitate the development of new GEMMs (including transgenic, knockout, conditional knockout and knock-in mice) using both conventional and CRISPR/Cas9-based technologies
  2. Provide a full range of support services to facilitate the use of these models, including breeding colony management, mouse line rederivation, embryo and sperm cryopreservation and cryorecovery
  3. Offer expert consultation on the development and use of GEMMs
  • Services

    Service

     

    Price

    (current as of 7/1/19)

    Generation of NEW GEMMs

    Transgenic Mouse Production

    • Purify transgene
    • Microinject into fertilized mouse eggs from desired genetic background and reimplant into foster females
    • Develop genotyping strategy and screen potential founder mice
    • A minimum of 3 founders usually generated

    $6,500

    (for production in CB6F1 background; additional

    cost for C57BL/6 or other background)

    KO/KI/CKO Mouse Production by CRISPR/Cas9

    • Design targeting strategy (overall model design; guide RNA selection; repair template selection)
    • Purchase gRNAs, templates

    $2,500

    (KO, small KI)

    $3,500

    (CKO, large KI)

    • Microinjection/electroporation of reagents into fertilized eggs of desired strain and reimplant into foster females
    • Develop genotyping strategies and screen potential founder mice; sequencing
    • Breed potential founders and screen offspring (including sequencing) to confirm germline transmission of desired allele

    $6,000

    (KO, small KI)

     

    $9,000

    (CKO, large KI)

    GEMM Support Services

    Colony Management

    • Acquire breeders (from JAX or other vendor, imported from another institution and rederived, or generated by TG/KO) and expand colony
    • Perform all mating, weaning, tail tissue collection, genotyping (usually by Transnetyx), record-keeping, and monitoring of colony
    • Transfer experimental animals as produced to investigator

    $55/hr

    (time in mouse room x 1.25)

    Genotyping

    • Send to Transnetyx (used for most routine genotyping)

    Transnetyx rate x 1.2

    • In-house genotyping

     

     

    $6/sample

    (DNA purif.)

    $7/sample

    (PCR)

    • Primer design, per assay

    $200/assay

    • Primer purchase

    $12/primer

    Mouse Line Rederivation

    • Facilitate imports with DAR
    • Expand colony if needed; purchase/transfer female breeders if needed
    • Superovulate females and mate to males
    • Collect fertilized eggs, transfer to MMRB, and reimplant into foster females
    • Screen offspring for desired allele
    • Transfer rederived mice to investigator

    $1,500

    In vitro Fertilization (IVF)

    • Superovulate (Hyperova) females and collect unfertilized eggs
    • Thaw or collect sperm and capacitate
    • Perform IVF and culture eggs overnight
    • Reimplant 2-cell embryos into foster females

    $1,500

    Sperm Cryopreservation

    • Collect and freeze sperm (from 2 males -> ~20 straws)
    • Thaw one straw for test of motility or fertilization
    • Store in LN2 (currently no cost)

    $600

    (swim test)

    $1,200

    (IVF test)

    Embryo Cryopreservation

    • Obtain male breeders from investigator
    • Purchase females or obtain from investigator
    • Superovulate females and mate to males
    • Collect fertilized embryos (morula stage) and freeze
    • Perform test-thaw of one straw
    • Store in LN2 (currently no cost)
    • A minimum of 200 embryos are cryopreserved

    $2,500

    Cryorecovery from Frozen Sperm

    • See IVF

    $1,500

    Cryorecovery from Frozen Eggs

    • Thaw straw and culture overnight (to blastocyst)
    • Reimplant into foster females

    $1,000

    MEF Production

    • Set up timed matings of investigator’s mice
    • Collect embryos from pregnant females at ~e13.5
    • Process embryos to create MEF cultures
    • Pass/split cells after 2 days
    • Genotype cells from each culture if from het x het matings
    • Transfer cultures to investigator

    $1,000

    (from homos)

     

    $1,250

    (from heteros)

    Management of IVIS Imager in MMRB Vivarium

    • Provide initial training to new users
    • Oversee sign-up for use through FORCE
    • Perform routine maintenance of facility; maintain O2 supply
    • Schedule annual maintenance

    $40/hr

    Consultation

    For all projects

    • Meetings to discuss experimental objectives, details of experimental design, optimal/alternative strategies, timeline, etc

    -----

    Other examples

    • Breeding and genotyping strategy consultation; genotyping trouble-shooting
    • Basic training on use of IVIS imager
    • Determining whether desired mouse lines are available from JAX or various consortia or repositories; completing MTAs
    • Facilitating import of lines from other institutions (in conjunction with DAR)
    • Provision of support letters for grant applications
    • Assisting with budget preparation for grants including GEMM production and breeding
    • Assisting with preparation of IACUC protocols

    -----

     Massey member subsidy benefit

    Massey Cancer Center subsidizes 25 percent of the cost of members’ core laboratory usage for the Transgenic/Knockout Mouse Facility Shared Resource.

    To receive this member benefit, please initiate core lab services using a VCU index code associated with your name. When you are billed for core laboratory usage, the FORCE system will automatically split the invoice between your index code and a Massey account established for this purpose. You will be charged 75 percent of the published rate and Massey will provide a 25 percent co-pay to cover the balance.

    Please direct any questions or concerns about the member subsidy benefit to Paul Fawcett, Ph.D., at pfawcett@vcu.edu or (804) 833-1959.

  • Equipment

    The Transgenic/Knockout Mouse facility is located on the 8th floor of the Molecular Medicine Research Building (MMRB). It occupies a 1,000-sq-ft suite that includes a 600-sq-ft laboratory, 150-sq-ft tissue culture room, and a 250-sq-ft animal holding room dedicated for mice related to GEMM production. Two additional 450-sq-ft holding rooms in MMRB are dedicated to housing mice in the breeding colony management program. A 150-sq-ft holding room in Sanger Hall is dedicated to holding mouse lines undergoing rederivation by the Transgenic/Knockout Mouse core.

    The laboratory is equipped with:

    • Olympus IX-71 inverted injection scope with stage cooler (for microinjection)
    • Two Eppendorf TransferMan 4r micromanipulators
    • Eppendorf Femtojet 4i microinjector
    • NEPAGENE NEPA21 electroporator
    • Olympus DP-72 Digital Camera
    • Vibroplane air table
    • Nikon SMZ-800 dual-head teaching microscope (for mouse surgery)
    • Nikon SMZ-645 microscope (for embryo manipulation)
    • Three Forma Scientific CO2incubators
    • Sutter P-87 pipette puller
    • DeFonbrune microforge
    • Bio-Cool III controlled rate freezer
    • Two VWR CryoPro LN2holding tanks
    • Two Thermo 8033 LN2holding tanks
    • Thermo LN2dry shipping unit
    • Marathon refrigerated centrifuge
    • Nikon Eclipse TS100 inverted scope (for cell culture)

    The molecular biology work associated with mouse production is carried out in Dr. Windle’s lab on the 7th floor of MMRB, which is fully equipped for molecular biological studies.

    The core also maintains an IVIS Spectrum II optical imager in the MMRB vivarium. Please contact Greg Campbell at gecampbell@vcu.edu or (804) 827-3974 for training and access to the imager.

  • Contact
    Greg Campbell; Jolene Windle, Ph.D.; Pam Watters, M.S.; Mark Subler, Ph.D.

     

    The Transgenic/Knockout Mouse Shared Resource is directed by Dr. Jolene Windle. Greg Campbell, the resource manager, has experience with all aspects of GEMM production and cryopreservation, and he oversees the IVIS Spectrum imager located in the MMRB vivarium. Dr. Mark Subler brings expertise in all aspects of GEMM-related molecular biology and is responsible for the resource’s genetic services, including transgene design and construction, CRISPR-Cas9 targeting strategy and reagent design, and genotyping. Pamela Watters has been involved in mouse husbandry for many years and is responsible for breeding colony management.

    Please contact Dr. Windle for new service requests.

 Citation

All publications that include results, services or products generated by the VCU Massey Cancer Center Shared Resources must include the following acknowledgement in the manuscript:

"Services in support of the research project were provided by the VCU Massey Cancer Center Transgenic/Knockout Mouse Core, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059."

PubMed Central (PMC) ID numbers are required for those publications that use CCSG-supported shared resources. All of Massey Cancer Center's shared resources are supported by the Cancer Center Support Grant.