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Transgenic and Knockout Mice Facility
http://med.stanford.edu/transgenic/
Facility Director: Yanru Chen-Tsai, PhD
Faculty Advisors: Michael Cleary, MD, and Dean Felsher, MD, PhD
Contact
Yanru Chen-Tsai, PhD
Director, Stanford Transgenic Research Facility
Room 3245B, CCSR Building
Stanford, CA 94305
Tel: 650-498-7604
Fax: 650-725-6902
Lab: Room 1210, CCSR Building
Stanford, CA 94305
Tel: 650-725-6871
Email: ychen@stanford.edu
Overview
The overall goals of the Transgenic and Knockout Mice Shared Resource are to provide high-quality embryo and stem cell manipulation, microinjection and related services to Stanford cancer investigators--in a timely manner and at a low cost--in support of their research needs. Investigators in eight programs of the Cancer Center employ genetically modified mouse models for their studies. For most cancers, the mouse is the most useful animal model that recapitulates the major features of human malignancies. Technological breakthroughs in the transgenic field allow for mouse genome manipulation, such as deleting, adding or mutating a specific gene or for inducible expression systems, providing powerful tools for in vivo studies of cancer causation and pathogenesis. Main services include:
- Consulting in all aspects of creating and characterizaing genetically modified mice
- Producing transgenic and knockout mice by microinjection
- Manipulating mouse embryonic stem cells for gene targeting
- Providing molecular and biological reagents associated with production of genetically modified mice
Services
Transgenic models
For transgenic models, we perform pronuclear microinjection with plasmid DNA fragments or BAC DNA. If needed, we also provide DNA purification services to ensure a higher success rate. For embryo donors, the available strains are FVB, C57BL7 and B6CBAF1.
Knockout models
We perform in vitro transfection of ES cells to obtain clones that have undergone homologous recombination. This is the crucial, highly demanding cell culture step necessary for introducing defined mutations into specific mouse genes. It is important that the ES cells remain pluripotent during culture and subsequent procedures to ensure germ-line transmission of the mutant allele. Following detailed screening procedures, generally performed by the investigator, to identify appropriately modified ES clones, the clones are microinjected into blastocysts to generate knockout mice.
Embryonic stem cells, mouse embryo fibroblast cells
and gene targeting vectors
To assist investigators who choose to perform various steps of the gene targeting protocol in their own laboratories, we provide a proven set of reagents that are useful for the production and analysis of knockout mice. These include low-passage ES cells with proven totipotency that are derived from the 129-strain background. We provide drug-resistant primary mouse embryo fibroblasts (MEFs) for use as feeder layers to maintain ES cells in an undifferentiated state during the selection of drug-resistant clones following gene targeting. Plasmids that contain various PGK-driven selectable markers for gene disruptions, LoxP and FRT sites and flanking polylinkers, as well as plasmids containing reporter genes, including lacZ and EGFP, are also available.
Cryopreservation, IVF and intracytoplasmic sperm injection (ICSI)
In addition to rederivation of frozen or fresh embryos, the shared resource also provides sperm freezing services for cryostorage of mouse lines. This technique, which is now preferable to embryo freezing, is important for preserving valuable lines while reducing labor and cage costs. IVF or ICSI is performed to resurrect mouse lines when such a request is made by the users.
Operation
The Transgenic and Knockout Mice Facility is located in the CCSR building room 1210 and RAF 025 and 027. The shared resource operates from 8:30 am to 5:30 pm, Monday through Friday, and is often operational Saturday and Sunday.
Procedures
Requests for services are routinely fielded by the facility director, Dr. Yanru Chen-Tsai, who discusses the proposed project with the user and provides guidance regarding potential strategies. To initiate services, users complete and submit an online requisition form at the facility’s web site. Forms are then added to the queue for services, and requests are processed in the order in which they are received.
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