| Summary: | <p>Abstract</p> <p>Background</p> <p>αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous <it>Cryaa </it>gene.</p> <p>Results</p> <p>Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse <it>Cryaa </it>gene. Two BAC transgenes, with EGFP insertions into the third coding exon of <it>Cryaa </it>gene, were created: the intact α<it>A-crystallin </it>148 kb BAC (αA-BAC) and αA-BAC(ΔDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the <it>Cryaa </it>gene in lens, but not outside of the lens. The number of cells expressing αA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of α<it>A-crystallin </it>locus derived from αA-BAC(ΔDCR3), 15 kb <it>Cryaa/EGFP</it>. A 15 kb region of <it>Cryaa/EGFP </it>supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of αA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing αA-crystallin in the lens pit.</p> <p>Conclusion</p> <p>We conclude that a 148 kb αA-BAC likely contains all of the regulatory regions required for αA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb <it>Cryaa/EGFP </it>region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular αA-crystallin expression. Finally, deletion of DCR3 in either αA-BAC(ΔDCR3) or <it>Cryaa </it>(15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.</p>
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