1. Establishment of monoclonal SMN2-GFP reporter line in HEK293.
Establishment of monoclonal SMN2-GFP reporter line in HEK293. (A) Representative images showing the monoclonal SMN2-GFP reporter line. Scale bar, 50 μm. (B) Southern blot showing expected GFP integration. All clones generate a 4,789-bp size DNA fragment comprising the GFP cassette after EcoRI and BamHI digestion. (C) Genomic DNA PCR analysis identifying correct GFP integration. (D) Western blot showing expression of SMNΔ7-GFP fusion proteins in all reporter lines generated. (E) Sequencing of the DNA sequences flanking the GFP cassette confirmed SMN2 gene rather than SMN1 gene integration. (F) GFP integration in SMN2 exon 8 showed normal splicing patterns as more than 90% SMN-Δ7 were spliced. (G) A small pool library screening identified 14 hits which brightened GFP fluorescence in the SMN2-GFP reporter line. Represented pictures before and after compound #8 (Z-FA-FMK) treatment are shown. Scale bar, 50 μm. (H) Quantification data showing that 14 hits significantly increased GFP intensity in the SMN2-GFP reporter cell line by more than 0.5-fold. Data were presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, as compared with DMSO group by two-tailed t test.
Yiran Wang et al. LSA 2019;2:e
© 2019 Wang et al.