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Title: Transcriptome perturbations within NIH3T3 cell lines expressing rhodopsin and its retinitis pigmentosa mutant and implication for drug screening      
keywords:
Transcriptome or Gene expression
ID:
PRJNA310601
description:
Mammalian cells are commonly employed to identify active compounds that could affect progression of many human diseases including retinitis pigmentosa. Here, using transcriptome analysis to compare NIH3T3 cells expressing rod opsin with the disease-causing a single point P23H mutation, we differentiated between genes affected by heterologous opsin expression and those influenced by the P23H opsin mutant gain- or loss-of-function. Surprisingly, heterologous expression of normal opsin causes changes in 783 out of 16888 protein coding transcripts more than 1 fragments per kilobase of transcript per million mapped reads (FPKM) in NIH3T3 cells despite that opsin is exogenous to this cell. The perturbed genes are involved in cell adhesion, morphology and migration and encode extracellular matrix proteins, growth factors, cytoskeleton proteins, glycoproteins or metalloproteases. Not fully overlapping 347 differentially expressed genes were affected when the P23H mutant opsin was expressed. Transcriptome perturbation by individual drug candidates also revealed that different active compounds can target distinct molecular pathways that result in a similar phenotype selected by a cell-based high throughput screen. This transcriptome approach is capable of detecting minute changes in the transcriptome and can be a key to therapeutic success of a candidate drug to restore the normal gene expression landscape in affected tissue. Overall design: Five cell assay conditions were arranged: 1) NIH3T3(GFP) cells were treated with 0.05% DMSO, labeled as WT; 2) NIH3T3(WT-opsin/GFP) cells were treated with 0.05% DMSO, labeled as OPSIN; 3) NIH3T3(P23H-opsin/GFP) cells were treated with 0.05% DMSO, labeled as P23H; 4) NIH3T3(P23H-opsin/GFP) cells were treated with 5 µM of compound 1 (an isoquinoline-2(3H)-hexanamide), labeled as T1; 5) NIH3T3(P23H-opsin/GFP) cells were treated with 10 µM of compound 2 (4-(5-chlorothiophen-2-yl)furan-2(5H)-one), labeled as T2. Each treatment condition was repeated in three 50 mm dishes as biological replicates. The medium was aspirated, and cells were washed with PBS after 24 h of each treatment before being lysed. Cells were lysed with TRIzol and total RNA was extracted from each sample according to the TRIzol reagent manual. High quality total RNA was subjected to sequencing library construction using 100 ng of total RNA as input. Transcript libraries were made with TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA). The 100 base paired-end sequenced samples were run on 2 lanes of a HiSeq 2500. Fastq files were generated from reads passing chastity filter.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA310601
authentication:
none
authorization:
none
ID:
pmid:27838510
name:
Mus musculus
ncbiID:
ncbitax:10090
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject