The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice

doi:10.1053/gast.2001.24846

Gastroenterology

Volume 120, Issue 7, June 2001, Pages 1720-1728

https://doi.org/10.1053/gast.2001.24846 Get rights and content

Abstract

Background & Aims: Keratins are intermediate filaments that are critical in cytoskeletal organization. Their roles in cellular processes are underscored by inherited human diseases in which germline mutations of keratins are found, as well as by transgenic and knockout mouse models that recapitulate those diseases. Keratin 19 (K19) has unique structural properties and developmental and spatial expression patterns. This suggests that K19 expression may correlate with important cell fate decisions in gastrointestinal tract epithelia. Methods: We used mouse K19 5' untranslated region and promoter sequences and fused it to the lacZ reporter gene in a transgene construct. Characterization was by β-galactosidase expression and X-gal histochemistry in gastrointestinal epithelia. Because endogenous K19 protein is transcriptionally regulated by the Kruppel-like transcription factor 4 (KLF4), we determined the spatial expression patterns of KLF4 and K19 in relationship to the lacZ reporter gene product. Results: K19-lacZ transgenic mice were found to have reporter gene expression in an epithelial-specific pattern. Expression was restricted to ductal epithelial cells in the pancreas, surface colonocytes, small intestinal villi, and gastric isthmus cells. Transgene expression correlated with K19 and KLF4 protein expression in the pancreas and stomach and was overlapping in the small and large intestine. Conclusions: The K19 promoter may be a useful tool to study epithelial cell biology and subsequent transdifferentiation programs, particularly the pancreas and stomach.

GASTROENTEROLOGY 2001;120:1720-1728

Section snippets

Generation of transgenic mice

The K19-lacZ vector contains a 2.1-kb BamHI genomic fragment of the 5' flanking region of the mouse K19 gene (gift of M. Lussier) linked to a 3.1-kb complementary DNA of the β-galactosidase reporter gene (Figure 1).

. Schematic diagram of K19-lacZ transgene construct.

DNA sequencing of the 2.1-kb 5' flanking region of K19 was performed (GenBank entry AF237661). The purified DNA was linearized by NotI restriction enzyme digestion, purified using Elutips, and used for microinjection. The DNA was

Generation of K19-lacZ transgenic mice and pancreatic ductal staining

Four founder lines were generated and genotyping was independently confirmed by PCR and Southern blot analysis. No differences in weight or physical appearance were noted between wild-type and transgenic mice. Age-matched transgenic and wild-type mice littermates were sacrificed between the ages of 3–4 months. No histologic abnormalities were noted in various tissues analyzed. Immunohistochemistry was performed with an antibody against β-galactosidase. Whereas no staining was observed in

Discussion

We have generated and characterized transgenic mice in which the K19 promoter, fused to the lacZ reporter gene, targets reporter gene expression in an epithelial-specific fashion. With respect to the gastrointestinal tract, there is expression in pancreatic ductal cells, the surface of colonocytes, villi of small intestine, and isthmus cells of the stomach, but none in the esophageal squamous epithelium. Why is there apparent dichotomy of expression between proliferative and differentiated

Acknowledgements

The authors thank members of the Rustgi laboratory (especially Dr. F. Stefan Schreiber), Dr. Gary Swain, Dr. Doris Stoffers, and Dr. Paolo Dotto for helpful discussions.

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^*: Address requests for reprints to: Anil K. Rustgi, M.D., 600A CRB, Division of Gastroenterology, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6144. e-mail: [email protected]; fax: (215) 573-5412.

^**: Supported by National Institutes of Health (NIH) grant R01 DK 53377 (to A.K.R.) and the Leonard and Madlyn Abramson Family Cancer Research Institute at the University of Pennsylvania Cancer Center (to A.K.R.), the Deutsche Forschungs Gemeinschaft Br 1806/1-1 grant (to F.H.B.), and the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases Morphology Core and Transgenic/Chimeric Mouse Core Facilities (P30 DK50306).

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Alimentary TractThe keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice*,**

Abstract

Section snippets

Generation of transgenic mice

Generation of K19-lacZ transgenic mice and pancreatic ductal staining

Discussion

Acknowledgements

Cell

J Mol Biol

J Biol Chem

J Biol Chem

J Biol Chem

Gene

Gene

Differentiation

J Biol Chem

Eur J Cancer Clin Oncol

J Invest Dermatol

J Biol Chem

Differentiation

Dev Biol

Differentiation

J Invest Dermatol

Patterns of expression and organization of cytokeratin intermediate filaments

Ann N Y Acad Sci

Intermediate filaments: structure, dynamics, function, and disease

Annu Rev Biochem

A structural scaffolding of intermediate filaments in health and disease

Science

The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14

J Cell Biol.

The cytoskeleton of digestive epithelia in health and disease

Am J Physiol Gastrointest Liver Physiol

Human type I cytokeratin genes are a compact cluster

Cytogenet Cell Genet

Arrangement of a cluster of three mouse type I keratin genes expressed sequentially during esophageal-type epithelial cell differentiation

Genomics

Alimentary Tract
The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice*,**