Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

Sabrina Roth,Patrick Franken,Riccardo Fodde,John Kong A San,Kim Monkhorst

doi:10.1186/1471-213x-12-18

Sabrina Roth, Patrick Franken + Show 3 more

Open Access

https://doi.org/10.1186/1471-213x-12-18

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Abstract

BackgroundTo facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein.ResultsFirst, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7–10 days.ConclusionsWe show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7–10 days.

Highlights

To facilitate the in vivo study of esophageal cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion
The luminal surface of the mouse esophagus is lined by a stratified squamous epithelium, which consists of distinct zones of cell proliferation and differentiation
Generation of transgenic ED-L2-rtTA mice Transgenic ED-L2-rtTA founder lines were generated with a construct consisting of the ED-L2 promoter cloned in front of the reverse tetracycline transactivator rtTA2-M2, previously described as an improved rtTA variant (‘Tet-On’) with strongly reduced background activity and a 10-fold increased sensitivity for doxycyclinedriven induction [10] (Figure 1A)

Summary

Introduction

To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. The slow cycling cells appear to preferentially cluster in the interpapillary zone and were proposed, progressive cell commitment within the differentiated zone This zone consists of multiple layers of progressively flattened and differentiated squamous cells which function as a protective barrier [4]. Differentiated cells above the basal layer can be visualized by cytokeratins 4 and 13 (CK4 and CK13) [3]

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