Services include:
- PCR, qPCR, RT-PCR;  Southern, Northern, Western, Southwestern blots

- CFTR/ENaC interactions , cell surface expression and membrane protein turnover/recycling

- Mucin biochemistry, Unfolded Protein Response (UPR) as inflammatory mechanism


Molecular Biology of Pulmonary Epithelium
The UNC CF Center has extensive capabilities in the molecular biology of human airway epithelia. With respect to the cell stocks mentioned, most stocks have been characterized for CFTR status and are available for other genetic studies. Specific molecular capabilities now include:
- Studies of gene expression in human pulmonary epithelia: Available are techniques and data that have emanated from extensive Affymetric analyses of whole genome expression in human airway and alveolar epithelia, and also more recent data generated from high throughput sequencing (454 pyrosequencing) of RNA to produce “RNA seq” libraries of human pulmonary epithelia under a variety of conditions.

- Gene transduction capacities in pulmonary epithelia: These capacities include the ability to express both conventional transgenes, siRNAs, and shRNAs in human pulmonary epithelia. The vectors of choice for these techniques include equine infectious anemia virus (EIAV) gene transfer and shRNA expression (see Figure 5), paramyxovirus (RSV/PIV3) vectors, AAV vectors, adenoviral vectors, and lipids/plasma formulations.

Airway Surface Liquid (ASL) depth
We have extensive experience in characterizing airway surface liquid physiology, in response to candidate therapeutic agents, under the “thin film” ASL technique. In “standard” versions of this technique, a combination of airway surface liquid (ASL) volume as measured by confocal techniques, indices of ion transport processes mediating ASL volume homeostasis from transepithelial microelectrode voltage measurements, coupled with micropipette liquid sampling for measurements of drug concentrations, can be made. Note, drugs can be added to the basolateral compartment of these preparations, the luminal compartment in perfluorocarbon, or indeed, more recently, by an interface to ultrasonic nebulizers that deliver aerosols to cultured airway surfaces at rates that mimic aerosol deposition in vivo.

Measurements of Mucin Secretion and Mucin/Mucus Function
We have extensive experience with cell culture and freshly excised murine tracheal models to measure baseline and regulated mucin secretion. These models are coupled to a spectrum of mucin measurement technologies, including antibody/ELISA based, biochemical/UPLC - light scattering technologies, and techniques that couple both technologies.

In addition, we have extensive experience in measuring biophysical properties of mucus that are important for drug penetration through mucus, mucus flow, and mucus adhesion.

Ciliary Beat Frequency
We offer an extensive array of technologies to measure ciliary function both in vitro and in vivo. Thus, we have available techniques for measurement of ciliary beat frequency in human and animal culture systems, measurements with high-speed videomicroscopy of ciliary beat form, ultrastructural measurement capacities to evaluate ciliary shaft structure, and the ability to perform proteomics of ciliary shaft proteins.

Additional Services
Leukocyte morphology, kinetics and effector function.
Mucociliary transport (MCT)

We offer numerous animal models of lung diseases, including the first “CF mouse” (UNCtm null). Current gene-targeted models include MUC1, 4, and 16 -/- mice, P2Y2-R -/- and P2Y4-R -/- mice, and pannexin -/- mice.In addition, the Marsico Lung Institute has the muco-obstructive βENaC transgenic mouse. βENaC mice have been crossed onto multiple strains and differing levels of transgene expression have developed. The mice exhibit a phenotype of airway surface liquid volume depletion, mucus hyperconcentration and adhesion, inflammation, early bacterial infection, airway re-modeling, and emphysema. βENaC mice have been used for multiple drug studies, including ENaC blockers, steroids, macrolides, and inhaled osmolytes.

- βENaC (Scnn1b) and ΔF508 CFTR transgenic mice – drug effects on lung and GI phenotype:  mucus burden/obstruction, inflammatory markers, microbiological evaluation.

- Mouse models of acute lung injury, sepsis, pneumonia/infection, cigarette smoke exposure, COPD.

- MCT and PFT in mice, mucus clearance in excised trachea.