Project description:Stargardt disease (STGD1), the most common inherited juvenile macular degeneration, is caused by biallelic mutations in the ABCA4 gene. Currently, there is no approved treatment. In this study, we investigated early-stage epigenomic changes in the retinal pigment epithelium (RPE) of Abca4-/- mice, a well-established model of STGD1. Reduced representation bisulfite sequencing (RRBS) revealed hypermethylation of gene regions associated with disease-related pathways, implicating methyl-CpG-binding protein 2 (MeCP2) and RE1-silencing transcription factor (REST) as potential regulators. Notably, DNA methylation of a subset of genes preceded their transcriptional change and disease phenotypes in Abca4-/- RPE. Together with the detected age-dependent increase in MeCP2 levels in Abca4-/- RPE, these findings suggest that early DNA methylation changes may contribute to RPE dysfunction and eventual cell loss in STGD1.
Project description:ABCA4 is an ATP-binding cassette transporter expressed in photoreceptor cells, where it plays a critical role in the visual cycle by facilitating the clearance of all-trans-retinal derivatives from photoreceptor outer segment discs. Mutations in ABCA4 are implicated in inherited retinal degenerative diseases such as Stargardt disease, leading to the accumulation of toxic bisretinoids and progressive photoreceptor and retinal pigment epithelium (RPE) dysfunction. Although the role of ABCA4 in the visual cycle is well established, emerging studies have begun to explore broader implications of ABCA4 dysfunction on retinal homeostasis and disease mechanisms. In the present study, we utilized Abca4⁻/⁻ mice to investigate the consequences of ABCA4 loss in photoreceptors and its impact on retinal structure and function using translating ribosome affinity purification (TRAP) followed by next-generation sequencing of photoreceptors RNA.
Project description:We report RNA-Seq analysis of the transcriptome of retinas and RPE/choroids from Abca4 knockout, Abca4 L541P;A1038V knockin and control wild type mice in order to better understand changes in gene regulation that could lead to retinal pathology in mice with ABCA4 deficiency/defect. Retinal and RPE/choroidal mRNA profiles of 30-day-old wild type (WT), Abca4-/- and Abca4L541P;A1038V/L541P;A1038V mice were generated by RNA-Seq, using Illumina Hiseq 2500
Project description:This study aims to investigate the impact of ISL1 overexpression on hPSC-derived islet differentiation. hPSCs from four genotypes (WT, ISL1-/-, PDX1-/-, and PAX6-/-) were differentiated to pancreatic progenitors (day 11), transduced with ISL1-IRES2-BFP or BFP control lentivirus, and further differentiated to SC-islet (day 18). The BFP+ cells were isolated by flow cytometry sorting and subjected to RNA-seq to assess transcriptional responses to ISL1 overexpression across different genetic backgrounds.
Project description:This study aims to investigate the impact of ISL1 overexpression on hPSC-derived islet differentiation. hPSCs from four genotypes (WT, ISL1-/-, PDX1-/-, and PAX6-/-) were differentiated to pancreatic progenitors (day 11), transduced with ISL1-IRES2-BFP or BFP control lentivirus, and further differentiated to SC-islet (day 18). The BFP+ cells were isolated by flow cytometry sorting and subjected to RNA-seq to assess transcriptional responses to ISL1 overexpression across different genetic backgrounds.
Project description:We report RNA-Seq analysis of the transcriptome of retinas and RPE/choroids from Abca4 knockout, Abca4 L541P;A1038V knockin and control wild type mice in order to better understand changes in gene regulation that could lead to retinal pathology in mice with ABCA4 deficiency/defect.
Project description:Purpose: Understanding the functional genomics of M.tb (Mycobacterium tuberculosis) and development of novel anti-M.tb drugs and vaccines needs an efficient gene edit tool. The aim of this study was to describe an easy and efficient gene edit tool for functional genomics study of M.tb. Method: A plasmid was designed containing mini CRISPR array and 400bp up and downside homologues DNA sequences from the target gene interposed by EGFP or BFP. This plasmid was transformed into M.tb that guided the endogenous CRISPR system of M.tb to cut the target gene and insert EGFP or BFP through homologues ends joining. The EGFP/BFP insertion was confirmed and the total genomic was extracted from mutated and wild type strains and subjected to High-throughput DNA sequencing. Results: The raw data was filtered by Trimmomatic and the clean reads were mapped to M.tb H37Ra reference genome with bwa 99.94%, and 99.97% genome of wag31 and esxQ deletion strains were covered respectively.
Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition.
