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Next Generation Sequencing Facilitates Quantitative Analysis of different developmental stage pEGFP-N1 injected zebrafish embryos and untreated group transcriptomes

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the early stage of pEGFP-N1 injected zebrafish embros transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: we injected the plasmid pEGFP-N1 into zebrafish zygotes by microinjection, and then when the embryos to develop to 1 hour-post-fertilization (hpf), 6 hpf and 12 hpf, these groups was collected respectively by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Bioinformatics analysis of the transcriptome sequencing found that the expression levels of genes related to the apoptosis process (Fig.1A), endogenous immune response and tumor necrosis factor-mediated signaling pathways were abnormal up-regulation, mainly including isg15, foxo3b, phlda3, cdkn1a, zgc:136826, and si:dkey-204l11.1, compared with the non-injected group at 6hpf. These genes are found in somatic cells to deal with foreign plasmids invasion.

ORGANISM(S): Danio rerio

PROVIDER: GSE165422 | GEO | 2022/06/15

REPOSITORIES: GEO

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Project description:Abstract：DNA transfection is widely used in biomedical research. However, transfection efficiency is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the possible involvement of the cGAS-STING signaling pathway, which induces type-I interferons in response to cytosolic DNA. We found deletion of cGAS enhances the expression of transfected genes. This enhancement is inversely correlated with the expression of type I interferons and interferon stimulated genes (ISGs). We provide evidence that the interferon responses can suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. As a result, administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression levels of exogenous genes in multiple cell lines and in primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression and this strategy may be applied to gene therapy. Goal：Analysis of mouse fibroblast cell line L929 transfected with pEGFP-N1 plasmid in the presence of DMSO or TBK1 inhibiter (BX795) . Results provide insight into molecular mechanisms underlying a inhibitor effect of BX795 on DNA-induced cGAS signaling which improves expression of transfected genes. Methods： L929 cells were pretreated with TBK1 inhibitor BX795 or mock treated with DMSO for 1 hour, then transfected with pEGFP-N1 plasmid. There are four groups in this experiment. S1, L929 cells mock-treated; S2, L929 cells transfected with pEGFP-N1 plasmid; S3, L929 treated with 0.5 uM BX795; S4, L929 treated with 0.5 uM BX795 and transfected with pEGFP-N1 plasmid. Each group has three replicates. There are total 12 samples in this experiment. Six hours later, total RNA of 12 samples was extracted using Trizol reagent from cells according to the manufacturer’s instruction. cDNA library construction and sequencing were performed by BGI Genomics (BGI-SHENZHEN, China) using BGISEQ-500 platform. High-quality reads were aligned to the Mus_musculus reference genome (Mus musculus GRCm38.p5) using Bowtie2 (version:2.2.5, parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200). The expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM) (version:1.2.12, default parameters). For the identification of differentially expressed genes (DEGs), we used PossionDis, which is developed by BGI Genomics, implementing the passion distribution method. We analyzed up-expression genes in S2 other than S1, S3 and S4.

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Next Generation Sequencing Facilitates Quantitative Analysis of different developmental stage pEGFP-N1 injected zebrafish embryos and untreated group transcriptomes

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