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. 2016 Feb 18;6:21307. doi: 10.1038/srep21307

Figure 3. Retinal immunostaining of SAMD7 and transcriptional activity of SAMD7 variants.

Figure 3

(a) Human retinal SAMD7 localization. Representative fluorescent images of horizontal cross-sections of human retina stained with anti-SAMD7 antibody (red, 1:250). Retinal counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (blue). SAMD7 immunoreactivity is predominantly detected in the photoreceptor nuclei, located in the ONL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (b) Luciferase experiments. Luciferase assays were performed in HEK cells, using different SAMD7 reporter constructs. The first construct consisted of CBR1 cloned in the pGL4.10 reporter vector containing a luciferase expressing gene without promoter. For the second construct CBR2 was cloned in the pGL3 promoter vector, upstream of a luciferase reporter gene and a minimal basal promoter. Finally, the third construct was obtained by cloning CBR2 upstream of CBR1 in the pGL4.10 vector. Two types of each construct were created, one containing the wt sequence of a healthy control, the other one consisting of the patient (pat) sequences containing the SAMD7 CBR variants. Cis-regulatory activity could be demonstrated for the CBR1 constructs, while for CBR2 only very little luciferase expression could be measured. In both cases, no significant difference in luciferase expression has been observed between patient and control. However, when combining CBR2 and CBR1, luciferase expression increases for the control construct, while for the patient construct there is a significant decrease in expression. *corresponds with p < 0.05 after a two-sample t-test. (c) Electroporation reporter assays in mouse retinal explants. As a confirmation for the luciferase experiments, electroporation assays were carried out for a CBR2/CBR1 construct in mouse retinal explants. Control and variant CBR2/CBR1 were cloned in a dsRed expressing vector without basal promoter and electroporated into isolated retinas of P0 mice. After eight days of in vitro culture, retinas were harvested, fixed and imaged, confirming that the combined cis-regulatory activity of CBR2/CBR1 is lost for the patient constructs.