Cancer Institute A national cancer institute
designated cancer center

Animal Tumor Models Shared Resource

http://med.stanford.edu/transgenic
http://med.stanford.edu/compmed/animal_care/

Contact

Vittorio Sebastiano, PhD
Instructor, Obstetrics and Gynecology
(650) 724-8084

Donna Bouley, DVM, PhD
Director, Animal Histopathology Lab
Research Animal Facility (RAF)
(650) 498-5403
Fax: (650) 725-0940

Overview

The Animal Tumor Models Shared Resource provides services in three major areas, including transgenic and knockout mice, preclinical oncology and animal histopathology services. The transgenic and knockout mouse production services were established in 1996 at the School of Medicine with the overall objective of providing genetically modified mouse models and supporting technologies to Stanford investigators at cost-effective rates. The Veterinary Service Center (VSC) in the Department of Comparative Medicine runs the animal histopathology service component of the Shared Resource. Major services include tumor analysis by histology, immunohistochemistry and pathological interpretation. The preclinical oncology services are new additions to the Animal Tumor Models Shared Resource. These services were funded and established by the Stanford Cancer Institute to support the development and testing of new compounds and to better meet the growing needs of the Cancer Institute members for preclinical animal models and therapeutics. The objectives of the Shared Resource are:

•   Consultation in all aspects of animal tumor models, including creation and analysis of tumor models and providing a database of tumor models in use by Stanford investigators

•   Production of transgenic and knockout mice by embryo and embryonic stem cell manipulation and microinjection

•   Full histopathology services for tissue processing, staining and pathological interpretation

•   Preclinical oncology services for tumor induction, drug administration, toxicity studies, data collection and analysis

Substantial cost savings, efficiencies and scientific advances accrue from providing these services through a centralized facility. The Shared Resource has generated over 500 transgenic and knockout mouse models in the last 5 years, each with multiple independent lines. Over 24,000 histology slides or tissue blocks were processed and over 670 pathology procedures were performed in 2008. The Shared Resource’s services were acknowledged or co-authored in hundreds of publications in peer-reviewed journals. In calendar year 2008, Cancer Institute members representing nine out of ten Programs utilized the Shared Resource. Future plans for the Shared Resource include implementation of tetraploid complementation to speed up knockout mouse production, expansion of the capacity of histopathology services, and offer services on complex tumor models such as orthotopic brain, prostate, pancreatic or renal tumor models. 

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, in addition to the common strains such as FVB and F1 hybrids, protocols have been developed for generating transgenic mice in strains such as C57BL/6, Balb/c, 129 and NOD, strains not normally amenable to embryo manipulation procedures and thus not commonly offered in equivalent facilities elsewhere. Even with added difficulties such as BAC transgenics and strains other than FVB and F1, we have achieved an overall average efficiency rate of 16% for transgenic mouse production, a rate that is on the high end of typical efficiency rates.

Knockout Models

The Facility performs in vitro transfection of embryonic stem (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 to identify appropriately modified ES clones, generally performed by the investigator, the clones are microinjected into blastocysts to generate knockout mice. By conducting all ES cell manipulation procedures in this Shared Resource, we are able to achieve a high success rate, a feature that attracts more users.

Cryopreservation, IVF and ICSI

In addition to rederivation of frozen or fresh embryos, our highly skilled research assistants perform embryo and sperm freezing services for cryostorage of mouse lines. These services offer investigators the ability to preserve valuable strains of mice that may be infected with murine pathogens or are difficult to breed, as may be the case with many transgenic or knockout mice.  These services also make it easier to transport strains of mice to other institutions, or to maintain a mouse strain without having to continue routine breeding.  In vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is performed to resurrect mouse lines when such a request is made by a user.

We anticipate a significant increase in demand for cryopreservation services once the Lokey Building is finished in July of 2010. All mice that are moving to the barrier facility in Lokey Building will need to be cryopreserved or rederived.

Preclinical Oncology Services

The preclinical oncology services provide in vivo tumor models and preclinical oncology trials for Cancer Institute investigators. We offer flexible and customized services to suit users’ research needs. Services include cancer cell culture, xenografts for tumor induction and teratoma induction from pluripotent stem cells, animal monitoring (blood draws, body weight and tumor measurements), drug dosing, toxicity studies, sample collection, histology, small animal imaging, and data collection and analysis. Details for these procedures are described below.

Cell and Tissue Culture

The Shared Resource maintains a panel of cryo-preserved human cancer cell lines that grow in immunocompromised mice. These cell lines include PC-3, MCF-7, MDA-MB-231, HCT, CWR22 and LNCaP. Investigators may provide us with a cell line of interest.

Tumor Induction

Animals are implanted subcutaneously or orthotopically into mammary fat pads with tumor cells. In some cases, the cells are implanted with 50% matrigel (BD biosciences) to promote uniform formation of tumors. We culture the cells and test for initial take rate in vivo to determine the number of cells needed for effective tumor formation (>80%).

Teratoma Formation Service

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells form teratomas with all three embryonic germ layer cells when xenografted into immunodeficient mice. Teratoma formation is especially important for assessing human ES and iPS cells since chimera and germline testing which is available for mouse stem cells is impossible for human cells.

Toxicity Study and Determination of Drug Doses

In case of a compound with known in vivo toxicity, pharmacokinetics and pharmacodynamics, we determine the formulation and dosages based on the literature. In case of novel compounds, we perform in vitro and in vivo preliminary toxicity studies and in vivo pharmacokinetic studies to determine appropriate dosage, frequency and route of administration.

Drug Administration

We administer test compounds topically, intra-peritoneally, intra-venously via tail vein, sub-cutaneously and orally using gavage or in the food or drinking water. We also use sub-cutaneous osmotic minipumps for consistent delivery of compounds with compatible solvents.

Data Collection

Results from tumor growth studies are interpreted and graph reports are generated using study management software specifically developed for oncology studies. Electronic calipers and scales are connected to the computers where the software is installed and with a click, tumor measurements and body weights of animals are entered into the computer. Excel files and graphs are created for the measurements and are sent to the investigators periodically. The data are regularly backed up onto our server.

Pathology Services and Animal Imaging

Tissues and blood samples are collected for histopathological analysis and are processed in the histology lab of the Shared Resource. We can also image the progression of a disease or trafficking of cells in vivo using bioluminescent and/or fluorescent imagers (for example, SAFI or IVIS imaging equipment) or in vitro (using fluorescent confocal microscope Cell Vizio) through the Cancer Imaging Shared Resource.

Furthermore, we provide consultation on all aspects of using animal tumor models, including study design, selection of an appropriate model, drug dosing strategy, data collection and analysis. We work closely with investigators and assist on animal use protocol preparation. This in-house resource of tumor models ensures maximum resource utilization available at Stanford with reliability. The tumor models we provide can be used for basic research to would advance our knowledge in cancer and will help expedite development of effective anti-cancer compounds.

Animal Tumor Model Database

With support from the Cancer Institute, a Research Specialist was hired to establish a mouse tumor model database. The database includes genetically modified mouse tumor models, including transgenic and knockout mice, that are produced or acquired by Stanford investigators. The purpose is to provide an animal model resource for Stanford mouse users and to share the available mouse models at Stanford. This database also provides a resource for the new animal preclinical oncology services. The web accessible database is fully established and currently contains more than 150 mouse models. New models are being added to the database on a daily basis.

Experimental Pathology and Histology

Histology Service

The Histology Service provides research quality histology, and uses routine paraffin processing, embedding, sectioning, and staining with standard histological methods including special orientation of tissues, serial and/or step sections and a range of immunocytochemical analyses.  It can process standard-sized sections as well as very large sections that require hand processing. The Histology Laboratory also routinely deals with frozen tissues (both fresh-frozen and fixed-frozen) and sections and stains them for a broad range of biochemical and immunohistochemical stains. Procedures are also available to quantify cellular apoptosis, angiogenesis, and cell proliferation.

Pathology Service

Certified veterinary pathologists of the Shared Resource provide full, customizable pathology services, including complete necropsy: gross examination of study animals, tissue collection and histopathological analysis of hematoxylin and eosin stained tissues from all major organs. Services are also provided in phenotypic analysis of genetically engineered mice; this characterization includes complete necropsy, complete blood count (CBC), and clinical chemistry panel. Researchers may also submit individual organs or tumors for histological evaluation. The veterinary pathologists provide training in basic necropsy and dissection methods, organ identification, techniques for tissue harvest and collection of body fluid, as well as training in perfusion fixation, immersion fixation, methods for OCT embedding and flash freezing. The necropsy laboratory is equipped with a down-draft necropsy table and chemical fume hood and is available at no charge for investigators to use by appointment. Pathologists can assist with interpretation of histochemical and immunohistochemical stains and electron micrographs and provide expertise in correlating pathologic changes with clinical signs and clinical diagnostic results. Pathologists can provide expertise on projects requiring unbiased stereology of tumor burden/size using Microbrightfield software, Neurolucida and Stereoinvestigator.

Other Services

To assist investigators who choose to perform various steps of animal model generation in their own laboratories, we provide a proven set of reagents. These include low passage ES cells with proven totipotency, drug-resistant primary mouse embryo fibroblasts (MEFs) for use as feeder layers to maintain ES cells in an undifferentiated state, tumor cell lines and a variety of DNA plasmids for construct building. We also provide staged embryos, timed-pregnant female mice and microinjection capillaries on request.

Operation

The Animal Tumor Models Shared Resource is located in the CCSR building of the Stanford University Medical Center and is operational from 8:30 am to 5:30 pm Monday through Friday, and sometimes on Saturday and Sunday.

A fully equipped wet laboratory for transgenic, knockout mouse production and preclinical oncology procedures is housed in the Center for Clinical Sciences Research (CCSR) building The microinjection laboratory is located in the Research Animal Facility (RAF), in the basement below Fairchild and Beckman Center Embryo, and sperm cryopreservation and derivation services are performed in a laboratory in the Edwards Building in room R322 or procedure rooms in RAF (AF038, AF025 and AF027). Histology services are available in the Edwards Building in rooms R330 and R310. The Pathology Laboratory is located in Research Animal Facility room AF064.

Procedures

Requests for services are routinely fielded by the Facility Director, Dr. Vittorio Sebastiano, who discusses the proposed project with the user and provides guidance regarding potential strategies. Please note the Transgenic Service Center will use iLabs for all scheduling and billing starting April 1, 2013. Please Register Here. Non-Stanford Researchers can register here External Researchers (iLabs works best with Mozilla Firefox)

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