Zebrafish lines and workflow. (A) A DJ-1 knockout line was previously established by using the CRISPR-cas9 method to target a 20 bp region of exon one of the park7 gene [19]. Lines expressing glial specific wild-type DJ-1 or DJ-1 c106a in a DJ-1 null background were constructed by using ISce1 transgenesis and regulatory elements of glial fibrillary acidic protein (GFAP). The viral 2A peptide allows expression of GFP and Flag-DJ1 as uncoupled protein. In the retina, the gfap promotor drives expression only in the Müller glia cells. (B) Glial expression of GFP in the Müller_DJ-1 line (b,d) versus KO_DJ-1 (a,c). Note that GFP expression was determined by using a GFP antibody, followed by a far-red secondary antibody to avoid interference from retinal autofluorescence. Prominent expression is found around the Müller cell bodies. GFP expression also extends through the Müller processes into the photoreceptor layer. Faint expression can be observed in the Müller foot processes on the inner limiting membrane. Bar, 50 μm. (C) A Western blot shows expression of endogenous DJ-1 and Flag-tagged DJ-1 from total brain extracts belonging to animals from which eyes were collected. Ponceau S staining was used as a loading control. Asterisk points to an unspecific band. (D) Sagittal view of the zebrafish eye and workflow employed in this study.
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