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T Li, L Lu, L Lu; Identification of Regulatory Element in Pax6 Gene Transcription . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3642.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of the present study is to investigate control elements in 5' flanking upstream of Pax6 gene for transcriptional regulation in the eye development. Methods: A 4.2 kb DNA fragment (P4.2) in the 5' flank region of pax6 gene upstream of initial part of intron 1 was isolated from mouse genomic DNA using PCR. P4.2 fragment and its 5'-end deletion mutants (P3.5, P2.3 and P1.2) were subcloned into the promoterless pßgal-Basic vector (Clontech). DNA constructs were introduced by electroporation into retinoblastoma (Rb) cells and rabbit corneal epithelial (RCE) cells. ß-galactosidase activity in transfected cells was analyzed using a luminescent ß-gal reporter system. In addition, the effect and tissue specificity of these constructs was confirmed in developing chicken embryos. Results: It has been shown that Pax6 transcription is initiated by two promoters, P0 and P1, and regulated by an enhancer located in the upstream of P0. We found that ß-galactosidase activity in non-transfected or promoterless ß-gal vector-tranfected Rb and RCE cells was barely detectable. There was strong increase in galactosidase activity in 3 days when these cells were transfected with P4.2-ß-gal construct. P4.2-ß-gal construct was injected into chicken embryo at the day of E1. The signal of X-gal staining was intensified in cornea, lens epithelium, neural retina and neurons of developing central nervous system from days of E3 to E8. In the retina, galactosidase expression appeared higher than in the cornea and lens. P3.5, a 5'-end deletion mutant of P4.2, did not affect the expression pattern. However, P2.3 mutant with further deletion significantly down-regulated ß-galactosidase activity in both cells and chicken embryo. Interestingly, P1.2, a deletion mutant of P2.3, restored ß-galactosidase expression in Rb and RCE cells. Conclusion: The control of Pax6 gene expression can be regulated by an element in the upstream of P0 promoter. This element may play a role as a repressor in the control of Pax6 transcription in the eye development.
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