Functional analysis of the promoter for the long isoform of collagen a1(IX) in ovo

Elena I. Frolova
UTMB
Dept. Microbiology and Immunology
301 University Blvd
Galveston, TX 77555-1019
telephone: (409)772-2373
fax: (409)772-5065
email: elfrolov@utmb.edu
prestype: Platform
presenter: Elena I. Frolova

Elena I. Frolova
Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas, USA

A great deal is known about the early stages of eye development, the differentiation of the lens and the formation of the neural retina. However, the differentiation of the anterior part of the optic cup into the ciliary body and the iris structures remains obscure. One of the few proteins known to be expressed specifically and in abundance in the prospective ciliary epithelium is the long-isoform of collagen a1(IX). Its transcript is detected in the nonpigmented layer of the ciliary epithelium even before any morphological changes can be observed. Therefore, an understanding of the transcriptional regulation of the long-isoform of the collagen a1(IX) gene will give insight into the transcriptional mechanisms regulating the early steps of ciliary epithelium differentiation. It was shown that correct differentiation of the ciliary epithelium requires the presence of the lens and probably the neural retina. This makes difficult to create cell cultures that precisely imitate the differentiating ciliary epithelium in vivo to use for transient expression assays in vitro. To overcome this problem we employed a new method, in ovo microelectroporation, for functional study of the long-isoform of the collagen a1(IX) promoter.

We used in ovo microelectroporation to introduce reporter plasmids containing different parts of the collagen a1(IX) promoter into cells of the optic cup in living chicken embryos. Promoter constructs were designed in which the promoter and part of the coding sequence for collagen a1(IX) gene were fused in frame with the coding sequence of green fluorescent protein (GFP). The GFP fusion constructs were then injected into the optic vesicle of chicken embryos. After electroporation, embryos were allowed to develop for 2-3 days. GFP expression was observed by confocal microscopy in whole mounts and in sections.

We demonstrated that the presence of intron 1 of the collagen a1(IX) gene is essential to direct its expression to the optic cup. Despite the difference in the size of intron 1 in chickens, mice and humans, this part of the collagen 1 (IX) gene contains several highly conserved sequences.

The advantages and disadvantages of using in ovo microelectroporation for functional study of the promoters will be discussed.