Abstract
Abstract: :
Purpose: VAL-opsin is a putative photoreceptive protein present both in a subset of retinal horizontal cells and diencephalic cells of adult zebrafish (Kojima et al., J. Neurosci. 20: 2845-2851, 2000), but the physiological role of VAL-opsin in these cells remains to be determined. The aim of this study is to identify the VAL-opsin-expressing cells in dissociated retinal cell cultures of zebrafish to use them for physiological experiments. Methods: Retinas were obtained from dark-adapted adult zebrafish and dissociated enzymatically and mechanically into isolated cells, which were then cultured in 70% L-15 medium. The cultured cells were fixed and stained with a VAL-opsin-specific antibody, VC. A 7.2 kb 5'-flanking sequence of VAL-opsin gene was isolated by screening a zebrafish genomic DNA library, and fused upstream of a GFP reporter gene to construct an expression vector, pVALuG7. Zebrafish embryos were injected with linealized pVALuG7 at the one or two cell stage. The injected embryos were raised to adults and tested for germ-line transmission of the transgene. Results: Consistent with previous work on zebrafish retinal sections, VC-immunoreactivity was found in a subset of horizontal cells 4h after dissociation of the retina, indicating the presence of VAL-opsin in these cells. However, the number of VC-immunoreactive horizontal cells significantly decreased by 24 h after dissociation. To facilitate identification of the VAL-opsin-expressing cells in "living" cell cultures, we have made transgenic fish that contain the VAL-opsin promoter linked to a GFP gene. Three stable lines of zebrafish bearing the transgene pVALuG7 in the germ cells have been established and are presently being studied. Conclusion: Retinal cell cultures made from pVALuG7-transgenic animals should be useful for physiological studies on the role of VAL-opsin in retinal horizontal cells.
Keywords: 497 opsins • 555 retina: distal(photoreceptors, horizontal cells, bipolar cells) • 606 transgenics/knock-outs