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Core B. Mouse Drug Screening and Transgenics Core.

Kanneboyina Nagaraju, PhD DVM

Margaret Sutherland, PhD

Yi-Wen Chen, PhD

Christopher Spurney, MD

Children’s National Medical Center, Washington DC

The overall goal of the Mouse Drug Screening and Transgenics Core is to provide centralized mouse model services to the Projects, and also to external investigators.  The only commercially available murine model of muscular dystrophy is the mdx mouse model (first defined as a true animal model of DMD by the PI of the Core in 1987; Hoffman et al. 1987).  The Core currently houses murine models for alpha, beta, and gamma sarcolgycan deficiency (limb-girdle muscular dystrophies), and will soon obtain nude/mdx mice from Hammersmith Hospital London commensurate with Dr. Partridge’s move to Washington DC.  Also, there are no previously existing means by which investigators can obtain a battery of functional and histological tests on mouse models of muscular dystrophy after experimental therapeutics treatment.

The three aims of the Core are as follows:

Specific Aim 1.  Maintain breeding colonies of mouse models of muscular dystrophy for use by Program investigators.  Mouse strains will include:  mdx, alpha sarcoglycan, beta sarcoglycan, gamma sarcoglycan, emerin EDMD, lamin A/C EDMD, calpain 3, dysferlin, and mdx/nude.  Mice will be sent to both Program investigators, and those outside the program requesting mice, at no charge.

Specific Aim 2.  Establish a functional testing facility for testing of therapeutic benefit in dystrophic mouse models.  This will include standardized histological assays (fibrosis, central nucleation, fiber size, Evans blue uptake), muscle functional testing (rotorod, wire hang test), cardiac imaging, and biochemistry (quantitative protein production).

Specific Aim 3.  Provide transgenic mouse production support for Program investigators.  This will initially focus on the needs of Project 1 (Dr. Lu) for oligonucleotide therapeutics.

Description of Functional Testing Facilities

1. Facility and support personnel: The mouse pre-clinical drug testing facility (DTF) is located at VA hospital across the road from Children’s National Medical Center. The VA animal research facility is accredited with AAALAC and is registered with the USDA and OLAW. The mice in DTF rooms are maintained under murine pathogen free barrier conditions in individually ventilated caging system with continuous health monitoring. Husbandry and manipulations are performed in accordance with IACUC approved standard operating procedures. Animals are maintained with ad-lib sterilized diet and sterilized water. Animals are group housed in solid bottom polypropylene cages with sterilized bedding in individually ventilated caging system. The facility is capable of maintaining and handling immunodeficient mice.  The facility has dedicated rooms for mouse procedures, motor activity monitoring, and muscle function testing.

Support personnel:

Director: Kanneboyina Nagaraju, DVM., Ph.D

Co-Investigator: Yi-Wen Chen, DVM., Ph.D

Co-Investigator: Christopher F. Spurney, MD

DTF Manager: Brandon Knight, BS

Technician/ Post-doctoral fellow: TBN

Animal facility staff for breeding, care and routine procedures (3 people)

2. Testing methods:

  • Muscle and motor function tests
    • Live animal:           
      • Rotarod
      • Treadmill
      • Grip strength
      • Wire hang test
    • Isolated mouse muscle: force  contractions and calcium measurements
  •  Imaging (cardiac imaging)
  •  Behavioral (open field digiscan)
  •  Biochemical
    • Hematology (white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, red cell indices (MCV, MCH, MCHC), and platelet count)
    • Chemistry (e.g., serum muscle enzymes  such as CPK, AST, ALT, LDH)
  •  Histological
    • H&E
    • EM
    • Special stains for muscle histology (Modified Gomori trichrome, Periodic acid-Schiff , Sudan Black, or oil red O, succinic dehydrogenase, NADPH, Acid Phosphatase, cytochrome oxidase, myofibrillar ATPase, )
  •  Immunological
    • Immunohistochemistry and immunofluorescence (T cells, B cells, Macrophages, dendritic cells, capillaries, complement components, cytokines and other study relevant stains)
    • Autoantibodies
    • Cytokines
    •  FACS
  • Genotyping of mouse strains by PCR and Southern blotting

3. Current capacity: 140 cages (700 mice)

4. Proposed muscular dystrophy models/strains

Duchenne dystrophy (mdx; mdx/nude immunodeficient)

Limb-girdle muscular dystrophy (a, b, and g sarcoglycan, calpain 3, dysferlin)

Emery Dreifuss Muscular dystrophy (emerin, laminin)

Inflammatory muscle diseases (MHC class I transgenic mouse model)

5. Costs of performing drug trial:

Please contact Dr. Nagaraju at knagaraju@cnmcresearch.org

 

Motor function tests:

-Rota-rod: Latency to fall

-Treadmill:

-Grip strength: Fore limb and hind limb strength

-Wire hang test: Latency to fall

  • Open field animal activity Monitor (Digiscan): It measures around 25 parameters including total distance moved, horizontal and vertical activities.

Imaging:

-Cardiac imaging:

  • Anatomical measurements: Left ventricular posterior wall and interventricular septum thickness in systole and diastole, left ventricular chamber size in systole and diastole, left ventricular length and volume in systole and diastole, aortic and pulmonary artery diameters
  • Functional measurements: Aortic and pulmonary outflow velocities and velocity time integrals, mitral and tricuspid e and a wave inflow velocities, heart rate, ejection time, isovolumic contraction and relaxation times, left ventricular mass
  • Functional calculations: Shortening fraction, ejection fraction, stroke volume, cardiac output, Tei index

-Magnetic resonance imaging (Coming soon):

  • To assess inflammation and fibrosis in skeletal muscle heart and diaphragm
            
Oligonucleotide anti-sense splicing strategies | Increased understanding of muscle stem cells  | Drug screening using both in vivo methods in mouse | High throughput in vitro assays | Murine drug screening and transgenic core
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