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Chick 1-Crystallin Enhancer Influences Mouse A-Crystallin Promoter Activity in Transgenic Mice

¹From the Department of Ophthalmology and the ⁴School of Medicine, University of Missouri, Columbia, Missouri; and the ³Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas.

PURPOSE. Both the –366/+43 and the –282/+43 mouse A-crystallin (or A) promoters have been effective at driving transgene expression in lens fiber cells, but not in lens epithelium. Because the chick 1-crystallin gene is expressed in lens epithelial cells, an enhancer was borrowed from this gene and linked to the A promoter. This heterogenic enhancer/promoter construct was tested in transgenic mice to see whether it was active in both lens epithelium and fiber cells while retaining lens specificity.

METHODS. The third intron of the chick 1-crystallin gene, which contains a lens enhancer element, was added to the 5' end of the mouse A promoter. We refer to this chimeric regulatory element as the enA promoter. To test its activity, we inserted coding sequences for five different genes. Transgenic mice were generated by pronuclear microinjection. Transgene expression patterns were analyzed by either X-gal staining, in situ hybridization or immunohistochemical staining.

RESULTS. When enA-lacZ transgenic embryos were stained with X-gal at embryonic day (E)11.5, ß-galactosidase activity was detected only in the eye. Histologic sections of the stained embryos revealed that lacZ was expressed exclusively in the lens, in both epithelial and fiber cells. Transgenic mice were also generated using either the original A- or the new enA promoter linked to an insulin cDNA. In situ hybridizations confirmed that the short A promoter targeted prenatal insulin expression specifically to the lens fiber cells, whereas the enA promoter was active in both lens epithelial and fiber cells. Developmental studies of the enA-insulin mice showed that the enA promoter became active at the lens pit stage and remained active in all lens cells, even at postnatal ages. The enA promoter also successfully directed expression of SV40 T-antigen (TAg), human E2F2, and dominant negative Sprouty2 (dn-Spry2) genes to lens epithelial and fiber cells. The lens specificity of the enA promoter was maintained in minigenes with different types of introns and polyadenylation signals.

CONCLUSIONS. A new lens-specific regulatory element was generated—the enA promoter, which can drive high levels of transgene expression in both lens epithelium and fiber cells throughout development. This modified promoter can be used for future transgenic studies of signal transduction and cell cycle regulation in lens epithelial cells.

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