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As Anne-Karina Perl, PhD, started her work on embryonic lung development, she established the pharmacokinetics of doxycycline application for the use of the tetracycline inducible lung specific gene expression formerly generated by Jay Tichelaar. She characterized in detail the onset and cellular localization of inducible gene expression during embryonic lung development (Perl et al.; Transgenic Res. 2002). The inducible system overcomes embryonic and adult lethality and systemic effects and opened the field to study any genes during embryonic development and in adult lung injury models. These transgenic mouse lines, SPCrtTA (SftpC rtTA line 1) and CCSPrtTA (Scgb1a1 line 1), are now used by more than a hundred researchers all over the world to activate genes in the lung epithelium of embryonic and adult mice.
To establish inducible tissue specific gene inactivation, Perl crossed the SPCrtTA activator line 1 with operator mice expressing the Cre recombinase and characterized extend and cellular localization of recombination events with the Z / AP reporter line, which expresses alkaline phosphatase (AP) only in cells after recombination (Fig.1) (Perl et al.; PNAS 2002).
The tetracycline inducible gene inactivation in the lung epithelium has since successfully been used to discover new aspects of many gene functions in lung development and lung maturation and is now widely used in murine lung research. We characterized recombination with the CCSPrtTA activator line 1 (Perl et al.; Am J Respir Cell Mol Biol. 2005). Using the CCSPrtTA line targets recombination to the trachea, bronchi, bronchioles and a subset of alveolar type II cells (Fig.2 and 3). GFP labeling of targeted cells revealed distinct cellular patterns in the trachea.
We recently finished the characterization of the CCSPrtTA activator line 2 (Perl et al. Am J Respir Crit Care Med. 2011). Useful comments about the strength and limitations of these mouse lines have been discussed in Perl AK, Zhang L, Whitsett JA, Am J Respir Cell Mol Biol. 2009, and in Whitsett JA, Perl AK. Am J Respir Cell Mol Biol. 2006.
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Lungs of triple transgenic mice
Left: Whole mount AP staining on an E13.5 lung from an SPCrtTA / tetO-Cre / ZAP embryo.
Middle: GFP fluorescence in trachea from an SPCrtTA / tetO-Cre / ZEG transgenic mouse on PN7. GFP positive cells are organized in a linear fashion along the trachea, and numbers of labeled cells increased from proximal to peripheral extrapulmonary airways. (lower left corner 5x magnification).
Right: Widespread GFP fluorescence was seen in lung parenchyma from an SPCrtTA / tetO-Cre / ZEG mouse on PN7.
Correlation of GFP with epithelial cell markers in the peripheral lung of CCSPrtTA/ tetO-Cre / ZEG transgenic mice
Dams were treated with doxycycline from E6.5 to PN7. At PN7, frozen sections of CCSPrtTA / tetO-Cre / ZEG lungs were stained for -tubulin, CCSP, pro-SPC or CGRP (red) and visualized for dual fluorescence with GFP (green). Some ciliated (-tubulin positive) cells expressed GFP. In the bronchioles, some Clara cells (CCSP positive) expressed GFP, however not all non-ciliated, GFP expressing cells expressed CCSP. A subset of alveolar type II cells (pro-SPC) expressed GFP. GFP was not detected in squamous type I epithelial cells. GFP expression (green) was not co-localized with CGRP.
Patterns of GFP labeling in the trachea
Dams were treated with doxycycline from E6.5 to PN7. Whole mount of the proximal trachea of a triple transgenic CCSPrtTA / tetO-Cre / ZEG mouse was visualized using an inverted microscope with fluorescence optics. Labeled cells were present in a dorsal ventral pattern with increased density of labeled cells overlaying the cartilage rings.
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