Resources

Transgenic and Gene Targeting Core

Message from the Director:

A new "Transgenic and Gene Targeting Core" has been established in the Charles C. Gates Regenerative Medicine and Stem Cell Biology Program at UCD. Our core facility replaces the Transgenic/Knockout Core that was part of the UCD Cancer Center. We are committed to provide the UCD research community with state-of-the-art services. Our goal is to assist PIs in designing and generating genetically engineered mouse lines to further biomedical research.

Our services include pronuclear injections (conventional and BAC transgenic mice), ES cell injections (e.g. knockout and knockin mice), cryo-preservation of embryos and embryo re-derivation (see below for a detailed

Embryonic stem Cell Iinjections (courtesy of Eppendorff Inc.)

list of services and prices). We also conduct gene targeting experiments. The PIs will provide the gene targeting construct and test ES cell clones for homologous recombination. We will conduct the entire cell culture work and inject recombinant ES cell clones into mouse embryos to generate chimeric mice. If requested, we will breed these chimeras to test for germline transmission of mutant alleles.

Pronuclear DNA Injection (Courtesy of Eppendorff Inc.)

We also provide consultation services for the UCD community. Usually, we discuss experimental design before we actually initiate the project. The goal is to ensure that the experimental strategy employed is likely to result in the successful generation of the desired mouse line. The initial consultation (usually a one hour meeting) is provided free of charge. Further consultations and assistance in designing the experimental strategy to generate and analyze mouse lines will be provided on a fee-for-service basis.

To streamline our communication with PIs, we have established an e-mail account that should be used to schedule services and to request consultations (makeamouse@ucdenver.edu). All core members have access to this e-mail account. To facilitate correct routing of your requests, please include the PI name and the requested service in the header of your e-mail (e.g. John Doe “schedule transgene injection"; John Doe “schedule consultation"). We will then route the request to the appropriate core member.

We are planning to introduce new services to accommodate the needs of our research community. This might include the generation of transgenic rats, and the use of retroviral vectors to generate transgenic mice. We will evaluate requests for services from the research community for new services on a regular basis.

We are also planning to provide you with transgene and gene targeting vector design and construction services in the near future. Please stay tuned. I will need your feedback with respect to the introduction of new services. Feel free to drop me an e-mail at makeamouse@ucdenver.edu

Lastly, our core services are partially supported by a grant from the SOM dean. We are planning to become self-sufficient within three years. This will not be possible without your support. Please use our services and provide us with feedback so that we can continue to improve our services and help you to achieve your research goals.

Peter J. Koch

Core Personnel:

Peter J. Koch, Ph.D.: Associate Professor of Dermatology
Director, Transgenic and Gene Targeting Core
Charles C. Gates Regenerative Medicine and Stem Cell Biology Program
University of Colorado Denver
(P) 303 724 0051
E-Mail: Peter.Koch@ucdenver.edu
Joseph Anderson, M.S., Laboratory Manager: Senior PRA
Embryo Manipulation Services
E-Mail: Joseph.Anderson@ucdenver.edu
Ling Wang, M.D.: PRA
Gene targeting in ES cells
E-Mail: Ling.Wang@ucdenver.edu
Wallace Chick, Ph.D.: Instructor
Transgene and Gene Targeting Vector Design
E-Mail: Wallace.Chick@ucdenver.edu
Shawna Johnson, B.S.: PRA
Breeding and maintenance of genetically engineered mouse lines
E-Mail: Shawna.Johnson@ucdenver.edu
Abby Knight, CVT, RLAT: PRA
Colony and Data Management
E-Mail: Abby.Knight@ucdenver.edu

To request or schedule services please send an e-mail to:
MAKEAMOUSE@ucdenver.edu
Please include the PI name and the type of service requested in the subject line.

Information and Protocols:

Producing Fertile Embryos for Cryopreservation and Rederivation: Information and requirements for producing embryo donors and breeder males for optimal embryo production for efficient cryopreservation and rederivation of investigator colonies.
DNA preparation for Microinjections
DNA preparation for Gene Targeting
BAC DNA Construct Preparation: Please contact Makeamouse@ucdenver.edu

Re-derivation and Cryopreservation Information

This information pertains to the TGTC standard rederivation and cryopreservation procedures and is not part of the VC sponsored rederivation project. These two procedures are separate. Crypreservation provides for long-term storage of mouse lines and rederivation is the means to regenerate these lines. Cryopreserved embryos can also be shipped directly to other institutions without the complicated regulations needed for live animal shipments.

Application Forms:

Note: Please use the latest versions of application forms from this list. Outdated forms will be returned to the investigator and resubmission on the current appropriate forms required.

Vice Chancellor's Cryopreservation and Rederivation into RC-2

Use for Vice Chancellor's rederivation project.

Standard TGTC Cryopreservation or Rederivation

The application allows separate selections of cryopreservation for storage and/or rederivation for colony speed expansion and rederivation without the need for prior cryopreservation.

DNA Constuct Microinjection

Your DNA construct will be validated for microinjection by our personnel and the application forwarded to TGTC for scheduling. The TGTC will inform you by email when your microinjections are scheduled.

Embryonic Stem Cell Services

Used to submit your DNA to the Cell Services Lab, where it will be processed for stem cell injections. Please follow the instructions on the form. Following completion of processing, you will be notified.

Stem Cell Injection

Please use this form to apply for ES cell injections. You will be notified when the injections are scheduled.

Please review this agreement prior to utilizing the services of the TGTC and the stem cell facility.

Approval to Generate Genetically Engineered Mice

Note: you must have an approved Production and Experimental IACUC protocol in order to produce transgenic animals.

Please visit the Institutional Animal Care and Use Committee (IACUC) web page for information regarding the approval of animal experimentation at UC Denver.

Transgenic & Gene Targeting Core Pricing, 2008-2009

Prices subject to change.

Gene Targeting Service	Price
Electroporation/Drug Selection (First 288 clones)	$2,863
Additional Clones (Per 96 well plate)	$600
Clone Expansion (Per Clone)	$371
Karyotyping (cost per clone) [mandatory before blastocyst injection]	$390
Removal of loxP- or FRT-flanked genomic sequences by transient transfection of ES cell clones with Cre expression plasmids or FLPE expression plasmids (contact us at makeamouse@ucdenver.edu for details)	$2,863
Gene Targeting Consultation with Peter Koch, PhD. (per hour)	$100

Chimeric Mouse Model Development	$1543
Listed price inlcludes Injections (~50 blasts minimum), minimum of three recipient females and embryo transfer surgeries. Additional recipients/surgeries extra.,
Embryo donor purchase and estimated per diems included in listed price. These charges billed separately through CCM.

Transgenic Mouse Model Development	$1693
Price includes construct microinjections, minimum of three recipient females and embryo transfer surgeries. Additional recipients/surgeries extra.
Embryo donor purchase and estimated per diems included in listed price. These charges billed separately through CCM.

Strain Rederivation (per line)	$1,765
Price includes minimum of three recipient females and embryo transfer surgeries. Additional recipients/surgeries extra.
45 day of animal per diems included (billed separately through OLAR).

Embryo Cryopreservation (per transgenic line)	$540
Line regeneration charges will be dependent upon the recipient female costs, embryo transfer surgery costs and facility per diems in effect on that date.

Miscellaneous Services
DNA Purification	$163
Pseudopregnant Recipient Females - each	$65
Embryo Transfer Surgeries - each	$106
Mating, Weaning, Tagging, Biopsies	$21
Weaning Only	$13
Repeat Biopsies	$26
Timed Pregnancies	$33
Miscellaneous Services hourly rate, (billed in 15 minute increments)	$35
PCR Genotyping of Mice (per 10 PCR reactions)	$33
Consultation with TGTC personnel (per hour)	$51

The Transgenic and Gene Targeting Core can test your chimeras or transgenic founder mice for germline transmission of the desired mutations. Please contact the Core (makeamouse@ucdenver.edu) for details. These experiments will be conducted in our own animal room, which is shielded from the general mouse population at CCM to ensure that your mice are efficiently protected from common mouse pathogens.
The prices listed above are charged to investigators affiliated with UCD who conduct non-commercial basic or clinical research. For prices charged to commercial entities, please contact us at makeamouse@ucdenver.edu

Note: To inquire about prices for scientist outside the UCD system or for scientists affiliated with commercial companies, please contact Peter.Koch@ucdenver.edu