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Secretion-Competent Mouse Tracheal Epithelial Cell Culture From the Genetically Altered Mouse*: Pathway Analysis Via Gene Array FREE TO VIEW

Linda D. Martin, PhD; Kenneth B. Adler, PhD; Nancy J. Akley, MS; Anne Crews, MS; Lioudmila Sharova, PhD
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*From North Carolina State University (Drs. Martin, Adler, and Mss. Akley and Crews), Raleigh, NC; and Virginia Tech (Dr. Sharova), Blacksburg, VA.

Correspondence to: Linda D. Martin, PhD, Assistant Professor of Cell Biology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St, Raleigh, NC 27606; e-mail: linda_martin@ncsu.edu

Chest. 2002;121(3_suppl):79S. doi:10.1378/chest.121.3_suppl.79S
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The ability to create knockout and transgenic mice with phenotypes mimicking a variety of lung diseases has led to a large body of knowledge detailing the role of various gene products in the development of these diseases. Similarly, the use of well-differentiated human airway epithelial cell cultures has led to an understanding of precise signaling pathways regulating cellular functions such as mucus secretion, adhesion molecule and cytokine expression, and epithelial cell proliferation. The ability to combine these two powerful research approaches lies with creating an in vitro mouse tracheal epithelial (MTE) cell culture system. Here, we report the development of such a primary cell system that maintains morphologic and functional characteristics of the in vivo mouse airway epithelium. Specifically, epithelial cells dissociated from intact mouse tracheas are grown in air/liquid interface culture in defined media with or without serum. Under both conditions, Alcian blue/periodic acid-Schiff–positive mucous cells are observed. In contrast, ciliary development appears to require serum, suggesting that it may be possible to further manipulate this cell culture system to allow precise study of either mucous or ciliated cell development. This cell culture system has been examined to ensure its epithelial nature as indicated by Western blot analyses showing the culture findings to be positive for cytokeratin 5 expression. Using a mouse mucin 5ac-specific antibody to detect secreted protein by enzyme-linked immunosorbent assay, the cultures are found to secrete mucin constitutively and in a stimulated manner in response to known secretagogues (phorbol 12-myristate 13-acetate and 8-Br-cyclic guanosine monophosphate). Although a single trachea yields only 1 cm2 of differentiated culture, our preliminary studies indicate sufficient material can be obtained to perform gene array analyses of control and interleukin-13–exposed MTE cell cultures. Thus, we anticipate use of the MTE cell culture system not only to determine specific signaling pathways important to airway epithelial cell changes during lung disease, but by employing cells from knockout and transgenic mice, we expect to obtain an understanding of how expression of genes controlling these pathways is altered by genetic changes. In this manner, it should be possible to directly interface in vitro experimentation to define precise signaling pathways in airway epithelial cells with in vivo whole animal studies.

Abbreviation: MTE = mouse tracheal epithelial

Supported by National Institutes of Health grants HL 66236 (Dr. Martin) and HL 36982 (Dr. Adler).




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