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.
Figure 5. Expertise in genetic manipulation of hTBE.
Top Panel: VSV-G pseudotyped pSLIK-based (G418 resistance) lentiviral vector was used express shRNA to the ENaC protein in an inducible manner [induced by doxycycline (Dox)]. Primary hTBE cells on plastic were infected with pSLIK lentivirus, selected with G418, subcultured at an air liquid interface to induce differentiation and induced with Dox.
A) > 50% mRNA reduction was seen +Dox compared to -Dox.
B) This corresponded to a >80% reduction in ENaC protein by western,
C) a corresponding reduction in ENaC function (amiloride) by Ussing chambers, and
D) an increase in airway surface liquid height representing decreased ENaC activity.
Bottom Panel: Inducible over-expression of red fluorescent protein (RFP) was obtained in a similar fashion using inducible lentiviral vector (pTRIPZ). The images show whole mounts stained with anti-beta tubulin IV for ciliated cells (green) and red fluorescence. RFP expression is robust and only in the presence of Dox (3D confocal stack).