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Robust Acinar Cell Transgene Expression of CreErT via BAC Recombineering

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Robust Acinar Cell Transgene Expression of CreErT via BAC Recombineering

Baoan Ji et al. Genesis.

Abstract

Pancreatic acinar cells are critical in gastrointestinal physiology and pancreatitis and may be involved in pancreatic cancer. Previously, a short rat pancreatic elastase promoter has been widely utilized to control acinar cell transgene expression. However, this partial sequence does not confer robust and stable expression. In this study, we tested the hypothesis that a transgene employing bacterial-artificial-chromosome (BAC) technology to express a tamoxifen-regulated Cre recombinase from a full-length mouse elastase gene (BAC-Ela-CreErT) would be more robust and stable. When founders were crossed with Rosa26 reporter mice nearly 100% of acini expressed beta-galactosidase after tamoxifen treatment. The expression was specific for pancreatic acinar cells and these characteristics have remained stable for 2 years. However, because of high levels of expression in differentiated acinar cells, this construct is tamoxifen independent in approximately 50% of adult acinar cells. This model of pancreatic acinar specific Cre expression is a powerful tool for future transgenic and knockout studies.

Figures

FIG. 1
FIG. 1
Gene expression driven by a short rat elastase enhancer was not efficient. (a) CreErT gene was inserted between a ~500 bp rat elastase enhancer and human growth hormone polyA signal. (b) Transgenic founders were crossed with Rosa26 reporter mice. Double transgenic mice were injected with tamoxifen at 3 mg/40 g body weight daily for 3 days and sections of the pancreata were stained with X-gal. Approximately 10–15% of acini were labeled in a mosaic pattern.
FIG. 2
FIG. 2
CreErT expression from a full length elastase gene promoter was highly efficient and specific. (a) CreErT gene was targeted between the last exon and polyA signal of the mouse elastase I gene in a bacterial artificial chromosome. (b). Founders were crossed with Rosa26 mice, injected with tamoxifen and stained with X-gal. Nearly 100% of acini were stained with no staining in ducts, islets, or blood vessels. (c) X-gal staining was negative in a centroacinar cell. (d). CreErT (red) was not colocalized with Hes1 (green), a centroacinar marker.
FIG. 3
FIG. 3
BAC-Ela-CreErT mice developed tamoxifen independence in acinar cells with high expression levels. Pancreas from a BAC-Ela-CreErT × Rosa26 embryo at 19.5 days post-coitus (dpc) was X-gal negative (a). Injection of pregnant mice with tamoxifen (3 daily injections with 1 mg/40 g) generated many X-gal positive cells in embryos removed at 19.5 dpc (b). In the absence of tamoxifen, the number of X-gal positive cells increased with age. Data shown are without tamoxifen on day 1 (c), day 7 (d), day 14 (e), day 29 (f), and day 60 (g) after birth. Injection of tamoxifen in animals after 8 weeks (three daily injections with 3 mg/40 g) led to the expected x-gal staining in nearly 100% of acini (h). Unstained cells are either islets (arrowhead) or ducts (arrow). Sections were counterstained with fast red. All were photographed at × 100 magnification. Quantitation of X-gal staining at the different times indicated the time-course of leakage of BAC-Ela-CreErT (i). The expression level of CreErT paralleled the level of endogenous elastase expression as indicated by Western blot (j).

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