Figure 1. Strategy to mimic CF-lung disease.
Dr. Wanda O’Neal, the Director of the Mouse Models Core Laboratory, is dedicated to in vivo studies involving transgenic and knock-out mice generated or procured through the CF Center Molecular Biology Core. Since most of the morbidity and mortality currently associated with cystic fibrosis is due the development of severe obstructive lung disease, an on-going interest of the Mouse Models Lab has been to develop and study mouse models of defective airway mucus clearance.
In vivo, effective mucus clearance depends on an integrated system comprising epithelial ion and water transport, mucin secretion, ciliary beat, phasic airway motion, and cough. Airway surface hydration is essential to maintain efficient mucus clearance, and is finely tuned by the balance between Na+ absorption (mediated by the epithelial Na+ channel, ENaC) and Cl- secretion (mediated by the Cystic Fibrosis Transmembrane resistance Regulator, CFTR, and the Ca++ Activated Chloride Channel, CaCC) across the airway epithelium. In Cystic Fibrosis, the absence of functional CFTR channels, ablates one of the egress pathway for Cl- and results in uncontrolled Na+ hyperabsorption that causes airway surface dehydration (Figure 1, left panel). After the discovery of the CFTR gene in 1989, several attempts had been made to generate a mouse model of CF by deleting or inactivating the CFTR gene in mice. However, the CF-like lung phenotype has proven difficult to recapitulate in vivo, likely due to differences in physiologically relevant Cl- secretory pathways between human and mouse airways. An alternative strategy to induce the pathologic imbalance between Na+ absorption and Cl- secretion that causes lung disease in CF airways, is to over-express ENaC in the airway epithelium, thus artificially increasing the rate of Na+ absorption (Figure 1, right panel). Indeed, this strategy has proven successful. In βENaC-transgenic (Scnn1b-Tg) mice, airway-targeted overexpression of the epithelial Na+ channel β subunit (βENaC, encoded by the Scnn1b gene) causes airway surface dehydration and results in airway mucus obstruction and inflammation that strikingly resemble CF lung pathology (Mall et al., 2004 and 2008). Airway inflammation is characterized by enlarged/highly vacuolated macrophages, chronic neutrophilia associated with elevated KC, MIP-2 and TNFα, and transient juvenile eosinophilia (Figure 2, A, B, C). Tracheal mucus obstruction is associated with neonatal mortality, which in congenic C57Bl/6N Scnn1b-Tg mice is limited to 20%. However, mucus plugging and mucous secretory cell metaplasia progressively extend into the intra-pulmonary bronchi (Figure 2, D and E) and higher levels of secreted mucins can be readily detected in bronchoalveolar lavage (BAL) from Scnn1b-Tg mice by western blot (Figure 2, F).
Figure 2. Lung pathology in congenic C57Bl6/N βENaC -Tg mice. A-B) Airway inflammation, H&E stain; C) Differential cell counts, longitudinal study; D) Mucus plugs and E) Mucous secretory cells (AB-PAS stain); and F) agarose western blot quantification of secreted mucins βENaC -Tg mice and WT littermates.
- Generation and characterization of congenic βENaC -Tg lines (in C57BL6N, C3H/HeN, BALBc/J, FVB/NJ, 129/Sv genetic backgrounds).
- Study of transgenic lines that overexpress different combinations of ENaC subunits (a and/or β and/or g).
- Study of transgenic lines that overexpress low amount of βENaC transgene (low-overexpressor line).
- Crossings of βENaC-Tg mice with selected gene-deleted mice (TNFa, IL-4Ra, MyD88, TLR2, TLR4, Muc1, Muc 4, Muc16, Muc5ac, Muc5b, CFTRDF508, NLRP3).
- Administration of pharmacologic treatments aimed at preventing and/or blunting the insurgence of obstructing lung pathology.
The Mouse Model Core Laboratory also provides support and expertise for other investigators in the CF Center regarding mouse colony maintenance, breeding strategies, mouse lung phenotyping protocols, submission of Applications to Use Live Vertebrate Animals, and compliance with IACUC guidelines.
Dr. Barbara Grubb, PhD, UNC Cystic Fibrosis Center.
Dr. Scott Randell, PhD, UNC Cystic Fibrosis Center.
Dr. Sherif Gabriel, PhD, UNC Cystic Fibrosis Center.
Dr. Carla Ribeiro, PhD, UNC Cystic Fibrosis Center.
Dr. Silvia Kreda, PhD, UNC Cystic Fibrosis Center.
Prof. Dr. Marcus A. Mall, University of Heidelberg (Germany).
Dr. Steve Murray, The Jackson Laboratories - Bar Harbor, ME.
Dr. Amit Gaggar,M.D., Ph.D., University of Alabama at Birmingham, AL.
Dr. Steven M. Rowe, M.D, M.S.P.H University of Alabama at Birmingham, AL.
Dr. Christopher M. Evans, Ph.D., MD Anderson Cancer Center, Huston, TX.
Prof. Dr. Sandra J. Gendler, Ph.D., Mayo Clinic in Arizona, Scottsdale, AZ.
The Mouse Models Core Laboratory is located on the sixth floor of the Thurston-Bowles Building. It is a combined 400 square foot office/laboratory space in Room 6029. Lab members have access to state-of-the-art personal computers for data analysis, data acquisition, and word processing. All computers are hardwired to the University network and to the Internet.
Mice are housed in the animal facility is located on the first floor of the Thurston-Bowles Building and can be entered by Medical School Access Card. These facilities are available to us through the University of North Carolina’s Division of Laboratory Animal Medicine, are AAALAC-accredited, and are veterinarian-supervised. The Thurston-Bowles DLAM facility is one of the newest at UNC-Chapel Hill. It is a 7570 square foot SPF (specific pathogen free) rodent facility servicing primarily Alcohol Studies, Cystic Fibrosis Center and Gene Therapy Center faculty. A total of 307 cage-spaces are currently available to us in Room 1132 of the Thurston-Bowles Animal Facility and, since the model was developed here, our colony of βENaC-Tg mice is thus far the biggest in the US.
The Mouse Models Core Laboratory is fully equipped with Nikon SMX 2B dissection microscope, Nikon Labophot transmitted light microscope, StatSpin Cytofuge2 cytospin centrifuge, and two Sorvall biofuge Fresco benchtop refrigerated centrifuges. One 4oC refrigerator and four -20oC freezers are available for storage of reagents and samples. The laboratory is also equipped with an AirClean600 PCR Workstation, three 2720 Applied Biosystem thermal cyclers (to run PCR genotying reactions), and six FisherBiotech electrophoresis apparatuses to perform agarose gel electrophoresis, for both DNA and mucus samples. In the laboratory we have also available an Eppendorf Biophotometer spectrophotometer, a Boekel Rocker II and a Boekel vacuum blotter system.
Mrs. Jane Kelly, BS, MS
Research Specialist, Laboratory Coordinator
Jane joined the Mouse Models Lab in 2003, and since then has been a pillar of the lab. She graduated from Salem College where she obtained a Bachelor’s of Science in Biology in 1996. She obtained her Master degree in Animal Science and Nutrition at NC State in 2003. She is Mouse Models Lab Laboratory Coordinator, as well as our lab manager. She supervises the mouse colony management and breeding strategies, trains our new personnel in genotyping and phenotyping protocols and actively participates in phenotyping efforts.
Mrs. Allison Volmer, BS
Allison joined the Mouse Models Lab in January of 2010. Allison graduated from Texas A&M University where she obtained a Bachelor’s of Science in Biomedical Science in 1995. Before moving to Chapel Hill, she worked at Baylor’s College of Medicine and The University of Texas- Medical Branch. She is responsible for colony management and genotyping of Mucin-deficient and TLRs deficient βENaC-Tg mice and she participates in phenotyping efforts.
Ms. Kristen J. Wilkinson
Kristen graduated from UNC in 2006 with a Bachelors of Science in Biology and a minor in Chemistry. She joined the Mouse Models Core in 2006 where she participated in mouse colony management, genotyping, and phenotyping of bENaC and Mucin deficient mice. In 2009 she left the core to pursue a Masters in Research degree in Biomedical Sciences with a concentration in Human Genetics from St. Georges, University of London. In 2010 she received the postgraduate award for the best overall achievement in her course. In August 2011 she returned to UNC and the Mouse Models Core as a Research Specialist.
The University of North Carolina at Chapel Hill
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Phone: (919) 843-1097
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