Abstract
The maturation of stratified squamous epithelium of the upper gastrointestinal tract is a highly ordered process of development and differentiation. Information on the molecular basis of this process is, however, limited. Here we report the identification of the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult mouse, Ada is highly expressed in the terminally differentiated epithelial layer of upper gastrointestinal tract tissues. The data reported here represent the identification and detailed analysis of a 1. 1-kilobase (kb) sequence located 3.4-kb upstream of the transcription initiation site of the murine Ada gene, which is sufficient to target cat reporter gene expression to the forestomach in transgenic mice. This 1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level comparable to the endogenous Ada gene. This expression is localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites.
Highlights
The mucosa of murine upper gastrointestinal tract tissues, i.e. tongue, esophagus, and forestomach, like the epidermis, undergoes organized progressive differentiation to form a mature stratified squamous epithelium, which is comprised almost exclusively of keratinocytes [1,2,3]
While the majority of research has focused on keratin genes involved in basal cell proliferation or early differentiation, and the early differentiation marker involucrin gene, only a few of the several genes involved in the latest stages of epithelial differentiation have been studied, including transglutaminase 3, loricrin [20, 23], and small proline-rich protein 1A [29]
Immunostaining analysis showed that ADA is localized predominantly to the granular layer of keratinized epithelium [32], rendering it another marker of the late stage of keratinocyte differentiation in the upper GI tract
Summary
Plasmid Construction—The 6.4CAT transgene, described by Winston et al [33] as the construct ADACAT, was subcloned into the BamHI site of Bluescript KSϩ II vector (Stratagene). 2.0FCAT, 1.1FCAT, 0.9FCAT, 0.6FCAT, and 0.5FCAT were generated by ligation of the pCAT construct to various restriction fragments from the 6.4-kb Ada flanking sequence. Transgenic Mice—Each transgene construct was restriction digested to remove vector sequence, isolated by agarose gel electrophoresis, and purified using the QIAquick Gel Extraction Kit (Qiagen Inc.). Genomic DNA was collected from tails of weaning pups and analyzed by Southern blot using a 1.4-kb cat-SV40 probe to identify transgenic mice [33]. For Southern blotting, 10 g of genomic DNA was digested with BamHI or NcoI, separated by agarose gel electrophoresis, transferred to NytranTM Plus membranes (Schleicher & Schuell), and hybridized according to the manufacturer’s instructions.
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