Embed Size (px)
Citation preview
Nat
iona
l Can
cer
Inst
itute
2015 CCR Mouse Models CourseMouse 101Laboratory Animal Sciences Program CORE Resources
2015 CCR Mouse Models CourseMouse 101Laboratory Animal Sciences Program CORE Resources
Rick Bedigian Ph.DInterim Director Laboratory Animal Sciences Program
October 19, 2015
Frederick National Laboratory
Laboratory Animal Sciences Program Laboratory Animal Sciences Program
CORE Resources
Centralized scientific resources have been established to provide investigators access to new technologies and to complement and advance NCI’s research efforts.
Criteria
• Institutional commitment • High demand • Current and developing technologies • Time savings • Cost effective
Strain Preservation
Histo-Pathology
Mouse Model Development
Animal CareAnd Technical Support
In vivo Imaging
NCI-Repository
LASP Core Programs
Germ FreeHigh Throughput Genotyping
Laboratory Animal Sciences Program Animal Program Management
LASP Frederick and Bethesda Facilities• Comprehensive management of animal facilities and operations
– Animal holding, husbandry, and veterinary care– Animal technical services comprising experimental procedures and
colony management– Quarantine of imported animals– Re-derivation of pathogen-carrying strains– Diagnostic testing services to maintain the health status and monitor
genetic integrity of animal research colonies
• Management of 24 rodent and nonhuman primate research facilities– Support of 251 principal investigators encompassing 630 active
Animal Study Proposals
Gene targeting by homologous recombination
Cryopreservation and assisted reproduction
Colony expansion of genetically engineered mice (GEM) for large-scale studies
Transgenic mice
pronuclear microinjectionwith
• Plasmid• Cosmid• Bacterial Artificial
Chromosome (BAC)• P1 Artificial
Chromosome (P1)• Lentiviral Vectors
Xenograft ModelDevelopment
• Orthotopic• Heterotopic
Inject foreign DNA into male pronucleus
Gene TargetingProduction of:• Knockin (Ki)• Conditional Ko and Ki
Screening for targeting events
Selection of Recombinants
Spermatozoa forin vitro fertilization
HS V-tk
Gene Xneo
WT ES4 ES2
Mouse Model Development
Contact :[email protected]
Frederick National Laboratory
Gene Editing Methodologies Gene Editing Methodologies• Gene editing is a type of genetic engineering that utilizes engineered nucleases to bring about
targeted alterations at specific sites within the DNA. Three gene editing methodologies presently being used include Zinc Finger Nucleases (ZFNs), Transcription activator-like effector (TALE) nucleases and CRISPER.
• The CRISPR genome editing system allows one to design sgRNA that targets a DNA sequence of interest. When expressed intracellularly in conjunction with the Cas9 endonuclease, the sgRNA directs Cas9 to the target sequence where it unwinds and cleaves the double-stranded. The advantages of the CRISPR/Cas9 genome editing system include the ability of editing multiple genes simultaneously, a simple and fast design process that does not require the reengineering of the nuclease for each target, and an editing efficiency that is similar or greater than ZFNs and TALENs.
• Ran, AE, et al., Nature protocols, Vol 8, no. 11, 2013:
Frederick National Laboratory
Cryopreservation Options Cryopreservation Options
Frederick National Laboratory
Colony Expansion, Strain Rescue, or Rederivation by In Vitro Fertilization (IVF):In vitro fertilization quickly produces large quantities of age-matched, genetically modified mice on a common inbred background. This technique is ideal for those strains where males have a short lifespan or strains with breeding difficulties. Embryos obtained can be washed and then transferred into pathogen-free recipients, resulting in specific, pathogen-free mice for research purposes.
Small Animal Imaging Program (SAIP)at Frederick National Laboratory
• Provide “state-of-the-art” in vivo imaging to the NCI community
• Ability to understand and develop new mouse models, molecular markers, and theragnostics, and monitor tumors in vivo
• Multiple ultrasound, nuclear, optical, and fluorescent options with corresponding data analyses
3T MRI(Philips Medical Systems)Normal Inflammation Polyps Detected Polyps Enlarge
NUCLEAR IMAGINGNanoSPECT/CT(Bioscan, Inc.)
OPTICAL IMAGINGBioluminescenceXenogen IVIS SPECTRUM (Caliper Life Sciences)
ULTRASOUND IMAGING(VisualSonics, Inc.)
Imaging angiogenesis using microbubbles tagged with VEGFR2 antibodies
Inveon PET/CT (Siemens Medical)
Tumor imaging using [18F]-deoxyglucose, a glucose analog, to image metabolic activity.
BiodistributionAnatomical and Physiological Volume Image Probe Uptake (Wash-In/Out) Parametric MapsKinetic ModelingFluorescence LifetimeTumor VasculatureBlood Flow
Data Analysis
FluorescenceMaestro-GNIR
(Cambridge Research Institute)
OPTIX MX2(Advanced Research Technologies)
Contact: [email protected]
Contact: [email protected]
Pathology/Histotechnology Lab (PHL)• Integrates anatomic pathology, molecular histology, and digital slide imaging• Works closely with other LASP and FNLCR cores to extend net capabilities• Website: http://web.ncifcrf.gov/rtp/lasp/phl/
Functional Units• Veterinary Pathology (Dr. Diana Haines, [email protected]• Histotechnology and Molecular (Dr. Larry Sternberg, [email protected])
• Services:– Histotechnology:
• Rodent necropsy and tissue stabilization • Hematology, clinical chemistry• Tissue processing, embedding,Tissue Micro-Array (TMA)• Microtomy, cryotomy, • Staining, molecular histology (IHC, ISH)• Target enrichment: laser cutting (LC), LCM, “macro” dissection • Nucleic acid Isolation, yield and quality assessments • Digital whole-slide scanning: bright field and fluorescent
– Veterinary Pathology• Animal health diagnostic support • Anatomic and toxicological pathology• GEM Phenotyping• ISH/IHC interpretation• Digital image annotation, analysis/scoring• LC/LCM/TMA support
Obtaining Genetically Engineered Mice (GEM)
Generation:• Institutional core facilities• Commercial production facility• Individual laboratories
Obtain already generated models:• Mutant mouse repositories-IMR, MMRRC, EMMA, NCI
Mouse Repository• Individual laboratories• KOMP
The NCI Repository
Resource generated in support of the NCI’s Mouse Models of Human Cancers Consortium (MMHCC) • Mission: Preservation, propagation, and distribution of
mouse models for cancer research
• Current Repository holdings– 137 strains being distributed
• AMDL performs genetic quality control for the MMHCC Repository
Laboratory Animal Sciences Program
http://web.ncifcrf.gov/rtp/lasp/intra/ • NCI Repository
http://mouse.ncifcrf.gov/
CCR Research Exchange (CREx) https://nci.assaydepot.com/users/sign_in#_animal-resources
International Mouse Strain Resources
The IMSR is a searchable online database of mouse strains and stocks available worldwide, including inbred, mutant, and genetically engineered mice. http://www.jax.org/resources/mouse_resources.html
MMRRC-Mouse Mutant Regional Resource Centers
http://mmrrc.org https://nci.assaydepot.com/pages/cores
Useful Website Links
