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. 2021 Sep 29;7(40):eabj3658. doi: 10.1126/sciadv.abj3658

Fig. 4. Cytoplasmic Alu cDNA is synthesized via retrotransposition-independent RT.

Fig. 4.

(A) Alu cDNA formation monitored by in situ hybridization of RPE cells of R. norvegicus (WT rat) and O. palustris (Oryzomys) transfected with Alu RNA. Green, Alu cDNA; red, SiR-actin; blue, DAPI. Scale bars, 10 μm. Representative of n = 6. (B) Quantitative PCR (qPCR) of Alu cDNA generated by endogenous RT activity in cytoplasmic fractions of RPE cells of WT rat and O. palustris incubated with Alu RNA. *P < 0.05, Mann-Whitney U test. (C) Quantification of Alu cDNA products in cytoplasmic fractions of O. palustris RPE cells by Alu-specific PCR as described above. Cells were transfected with a control plasmid (pEmpty) or plasmids to enforce expression of rat L1 ORF1p, ORF2p (in the presence or absence of EFV), EN-deficient (EN−) ORF2p, or RT-deficient (RT−) ORF2p. *P < 0.05, Mann-Whitney U test. The error bars in (B) and (C) represent the means ± SEM. (D and E) Fundus photographs (left) and corresponding representative RPE sheet micrographs stained for ZO-1 (red) (right) of WT rat and O. palustris. Scale bars, 10 μm. Arrowheads in fundus images denote the boundaries of RPE hypopigmentation. Binary and morphometric quantification of RPE degeneration are shown. *P < 0.05; **P < 0.01; ***P < 0.001, Fisher’s exact test for binary; two-tailed t test for morphometry. PM, polymegethism [mean (SEM)]. n = 6 to 12. (D) RPE morphology in WT rats and Oryzomys after administration of Alu cDNA or mock injection. (E) RPE morphology in Oryzomys after Alu RNA administration following enforced expression of EN- or RT-deficient L1 ORF2p.