Mock: treated with Lipofectamine 2000 only; no treatment: no transfection.(PDF) pone.0197373.s001.pdf (35K) GUID:?DDE00973-8C8C-44AC-9B4D-DBE55F31E357 S2 Fig: The expression of EGFP- and TagRFP-conjugated proteins and TagBFP proteins in the reporter cell collection. EGFP: green fluorescence images using the (Ex lover/Em = 470/525 nm) filter, and TagRFP: reddish fluorescence images using the (Ex lover/Em = 545/605 nm) filter. Mock: treated with Lipofectamine 2000 only. The analysis was duplicated and repeated five instances to ensure the results were reproducible.(PDF) pone.0197373.s002.pdf (2.1M) GUID:?9B9E79B1-9167-430E-ABAC-F3DE5AB709F0 S3 Fig: Scatter-plot of the reporter cells after SSO transfection by HCS. Reporter cells seeded on 96-well black plates were transfected with the indicated SSOs at 100 nM. Twenty-four hours after transfection, fluorescence images of the reporter cell collection were acquired using ToxInsight. The captured fluorescence images were analyzed using the Thermo Scientific Cellomics Spot Detector V4 system, to obtain scatter plots of all solitary cells in each well. The X axis shows the total intensity of EGFP-conjugated proteins in each cell, and the Y axis shows the total intensity of TagRFP-conjugated proteins in each cell. The analysis was duplicated and repeated five instances to ensure the results were reproducible.(PDF) pone.0197373.s003.pdf (120K) GUID:?9C21DF8B-BA10-49BB-9F0F-70048A32F862 S4 Fig: Exon skipping activity of 3-mix LNA-based SSO cocktails using the established reporter cell line. Reporter cells were transfected with the indicated SSOs at 100 nM and incubated for 24 h. The % exon 51 skipping was determined as the amount of exon skipped transcript relative to the total amount of exon skipped plus full-length transcripts. Ideals represent the imply standard deviation of three self-employed experiments performed in duplicate.(PDF) pone.0197373.s004.pdf (27K) GUID:?0D2DC4FE-A031-468D-B5CB-65B68DF089C4 S5 Fig: Estimation of splice factor binding sites in the human being exon 51. Potential exonic splicing enhancer (ESE) sites of splice factors SRSF1, SRSF1 (IgM-BRCA1), SRSF2, SRSF5, and SRSF6 in human being exon 51 (including 50 bp of the flanking intronic sequence). These ESE sites are expected by ESE finder 3.0 . The expected ESE sequences are candidate SSO target sites for inducing exon skipping.(PDF) pone.0197373.s005.pdf (89K) GUID:?30147F8A-9A0E-4FB5-8BF2-199C9D73A80D S1 Table: SSOs utilized for the assay. Twenty-one LNA-based SSOs, PRO-051, and AVI-4658 for dystrophin exon 51 skipping are demonstrated. Sequences are demonstrated from 5 to 3. Capital characters with (L); LNA. Small characters: DNA. Capital characters with (M); 2-OMe RNA. Capital characters with (P); PMO. ^; phosphorothioate backbone. For assay systems that allow for GSK189254A the quick and simple testing of SSOs are essential for optimizing SSO design. In this study, we founded a novel tri-chromatic reporter cell collection for SSO testing. This reporter cell collection is designed to communicate three different fluorescent proteins (blue, green, and reddish) and was employed for high content material screening (HCS, also known as high content material analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the manifestation levels of fluorescent proteins. The blue fluorescent protein is stably indicated throughout GSK189254A the cell and is useful for data normalization using cell figures. Furthermore, both the green and reddish fluorescent proteins were utilized for monitoring the splicing patterns of Rabbit polyclonal to Acinus target genes. Indeed, we shown that this novel reporter cell collection involving HCS prospects to a more quick and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief testing of Locked nucleic acids (LNA)-centered SSOs focusing on exon 51 in was performed using the reporter cell collection. The LNA-based SSO cocktail shows high exon 51 skipping inside a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous mRNA in human being rhabdomyosarcoma cells. Intro Antisense-mediated splicing modulation is an attractive therapeutic approach for many genetic disorders including RNA mis-splicing . A earlier study exposed that over 60% of point mutations result in splicing errors . Moreover, in 2016, the US Food and Drug Administration (FDA) authorized two splice-switching oligonucleotides (SSOs): eteplirsen for the Duchenne muscular dystrophy (DMD) and nusinersen for spinal muscular atrophy (SMA) . Therefore, SSOs represent superb candidates for the further development of medical therapies for genetic disorders. Locked nucleic acids (LNA), also known as 2-mouse myotubes . Moreover, gene show an increased splicing modulatory effect . To develop novel antisense-based medicines, we and additional organizations possess previously reported the optimization of SSO design e.g., target sites, guanine-cytosine (GC) content material, melting temp (testing systems for the quick and simple evaluation GSK189254A of exon skipping activity are GSK189254A important because of the need to design and compare many candidate SSOs. Until now, GSK189254A many studies possess focused on reporter systems for the quick testing of pre-mRNA splicing [17C25]. Amongst them, exon 51 using SSOs and HCS. Moreover, we performed a brief testing of LNA-based SSOs.
- Genes Analyzed We analyzed the expression level of genes previously reported as related to the following processes or pathways: JAs biosynthesis (and [33,70]), JA signaling (and ), MBW complex (and repressor ), and anthocyanin and PA biosynthetic genes ([15,71,72], Supplementary Table S10)
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