In vitro and in vivo tracing of fluorescently tagged cell types in lung

Background:

Lung development process encompass various cellular processes including cellular interactions like alveolarisation, septa formation, differentiation and trans differentiation processes. Similar to this, some lung diseases are characterized by tissue remodeling processes. The lung is a complex organ with various cell types (e.g. alveolar epithelial type I and type II, fibroblast, endothelial and smooth muscle cells). There are specific markers to distinguish these cells but many of these markers are intracellularly expressed which limits their isolation and manipulation at transcriptional or translational level. To visualize the trans differentiation processes either in vitro or in vivo, we have developed the strategy to create a construct labeling 2–3 cell types either under the control of specific promoters or the use of CRE/LOX and FLP/FRT system to permanently label cell types in case of trans differentiation of the cell from one lineage to another.

Methods:

Cloning, cell culture, cell transfection, generation of transgenic mice

Results:

We have generated different plasmids expressing either one or two colours under specific promoters. Additionally, we have created a single plasmid with three cell type specific promoters driving expression of three different fluorescent molecules. These constructs are validated in cell culture for the expression of the fluorescent proteins in the respective cell types and given for microinjection to generate transgenic mice. With these constructs we cannot track the transdifferentiation process from one cell type to another. To overcome this limitation, we are creating another plasmid having CRE/LOX and FRT/FLP based system to label the cells permanently.

Outlook:

The creation of transgenic mice with 2–3 cell specific labeling will facilitate cell specific sorting, in vivo imaging and stereological quantification with regard to scientific questions in lung development and diseases.