Transgenic Knockout Mouse Shared Resource - VCU Massey Comprehensive Cancer Center

About the Transgenic/Knockout Mouse Shared Resource (TGKO)

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 TGKO are to:

Facilitate the development of new GEMMs (including transgenic, knockout, conditional knockout and knock-in mice) using both conventional and CRISPR/Cas9-based technologies
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
Offer expert consultation on the development and use of GEMMs

Services

Service		Price
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	$7,000 (for production in C57BL/6J or CB6F1 background; additional cost for other backgrounds)
KO/KI/CKO Mouse Production by CRISPR/Cas9	Design targeting strategy (overall model design; guide RNA selection; repair template selection) Purchase gRNAs, templates	$1,500 (KO, small KI)
		$3,000 (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	$6,500 (KO, small KI)
		$10,500 (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	$62/hr (time in mouse room x 1.5)
Genotyping	Send to Transnetyx (tailed by Shared Resource)	Transnetyx rate x 1.2
	Send to Transnetyx (tailed by Investigator)	Transnetyx rate
	In-house genotyping	$7.50/sample (DNA purif.) $9.00/sample (PCR)
	Primer design, per assay	$275/assay
	Primer purchase	$14/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 Alternative method: see IVF	$1,800
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	$1,800
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)	$800 (swim test) $1,500 (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	$2,600
Cryorecovery from Frozen Sperm	See IVF	$1,800
Cryorecovery from Embryos	Thaw straw and culture overnight (to blastocyst) Reimplant into foster females	$1,250
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	$1,500 (from homos) $2,000 (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 subsidizes 25 percent of the cost of members’ core laboratory usage for the following Massey-supported, fee-for-service shared resources:

Flow Cytometry Shared Resource
Lipidomics & Metabolomics Shared Resource
Microscopy Shared Resource
Tissue and Data Acquisition and Analysis Core
Transgenic/Knockout Mouse Shared Resource
Cancer Mouse Models Core
Proteomics 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 Rams 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 paul.fawcett@vcuhealth.org or (804) 827-0975.

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.

Jolene Windle, Ph.D.
Resource director
(804) 828-5843
jolene.windle@vcuhealth.org

Greg Campbell
Resource manager
(804) 827-3974
gecampbell@vcu.edu

Pamela Watters
Breeding colony manager
(804) 628-3104
pweller@vcu.edu

Mark Subler, Ph.D.
Genetic services manager
(804) 828-8325
mark.subler@vcuhealth.org

Service		Price
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	$7,000 (for production in C57BL/6J or CB6F1 background; additional cost for other backgrounds)
KO/KI/CKO Mouse Production by CRISPR/Cas9	Design targeting strategy (overall model design; guide RNA selection; repair template selection) Purchase gRNAs, templates	$1,500 (KO, small KI)
		$3,000 (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	$6,500 (KO, small KI)
		$10,500 (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	$62/hr (time in mouse room x 1.5)
Genotyping	Send to Transnetyx (tailed by Shared Resource)	Transnetyx rate x 1.2
	Send to Transnetyx (tailed by Investigator)	Transnetyx rate
	In-house genotyping	$7.50/sample (DNA purif.) $9.00/sample (PCR)
	Primer design, per assay	$275/assay
	Primer purchase	$14/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 Alternative method: see IVF	$1,800
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	$1,800
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)	$800 (swim test) $1,500 (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	$2,600
Cryorecovery from Frozen Sperm	See IVF	$1,800
Cryorecovery from Embryos	Thaw straw and culture overnight (to blastocyst) Reimplant into foster females	$1,250
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	$1,500 (from homos) $2,000 (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 subsidizes 25 percent of the cost of members’ core laboratory usage for the following Massey-supported, fee-for-service shared resources:

Flow Cytometry Shared Resource
Lipidomics & Metabolomics Shared Resource
Microscopy Shared Resource
Tissue and Data Acquisition and Analysis Core
Transgenic/Knockout Mouse Shared Resource
Cancer Mouse Models Core
Proteomics Shared Resource

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.

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.

Jolene Windle, Ph.D.
Resource director
(804) 828-5843
jolene.windle@vcuhealth.org

Greg Campbell
Resource manager
(804) 827-3974
gecampbell@vcu.edu

Pamela Watters
Breeding colony manager
(804) 628-3104
pweller@vcu.edu

Mark Subler, Ph.D.
Genetic services manager
(804) 828-8325
mark.subler@vcuhealth.org

Acknowledge the Transgenic Knock-Out Mouse Shared Resource

All publications that include results, services or products generated by the VCU Massey Comprehensive 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 Comprehensive Cancer Center Transgenic/Knockout Mouse Shared Resource, 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 VCU Massey Comprehensive Cancer Center's Shared Resources are supported by the CCSG.

This Shared Resource is jointly supported by VCU Massey Comprehensive Cancer Center and the Office of the Vice President for Research and Innovation.