N39 APOA1-tdTomato drug screen
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1142560 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30057600.fastq.gz | Fastqsanger.gz | |||
| SRR30057601.fastq.gz | Fastqsanger.gz | |||
| SRR30057602.fastq.gz | Fastqsanger.gz | |||
| SRR30057603.fastq.gz | Fastqsanger.gz | |||
| SRR30057604.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: Comparision transcriptomes of isolated tdTomato+ and tdTomato- OLs from the brains and spinal cords of infected mice at 30 dpi to those of OLs isolated from mock-infected central nervous system (CNS) samples by nextgen sequencing Methods: Brains and spinal cords were harvested at day 30 p.i. and processed as described above. After Live/Dead staining and treatment with Fc block, OLs were identified using APC-anti-O4 (1:50; Miltenyi). RNA was extracted from sorted OLs using an RNeasy Micro Kit (Qiagen). RNA sequence analysis was performed at the University of Minnesota Genomics Center. Results: We identified 158 and 115 genes, that showed a significant difference (p<0.05) when tdTomato+ and tdTomato- cells were compared to cells from mock-infected mice, respectively, with 61 mRNAs common to both tdTomato- and tdTomato+ cells
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Project description:The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain excitatory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:CamKIIα-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:CamKIIα-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and excitatory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.
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Project description:The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain inhibitory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:Slc32a1-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:Slc332a1-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and inhibitory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.
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Project description:Background: Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit in increasing production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance, and other genetic diseases, suggesting that they could be used to identify novel drivers of C7 production. Methods: A keratinocyte C7 reporter cell line was created by integrating a tdTomato fluorescent marker into the last exon of the endogenous COL7A1 gene. A genome-wide CRISPR activation (CRISPRa) screen was performed with the C7_tdTomato reporter to identify genes and pathways that increase C7 expression. High tdTomato-expressing cells were sorted and sequenced to identify the single guide RNAs (sgRNAs) that became enriched relative to the starting material. Pathway analysis was performed on the corresponding genes to identify regulators and pathways that influence C7 expression. A targeted drug screen was performed in three different keratinocyte cell lines based on the CRISPRa screen results, and C7 upregulation was evaluated. Results: The C7_tdTomato cell line was validated as an effective reporter cell line for detection of C7 upregulation. The CRISPRa screen identified two genes, DENND4B and TYROBP as top hits based on enrichment in the high fluorescence population and representation with multiple sgRNAs. Pathway analysis of the CRISPRa screen showed enrichment of toll-like receptor and interferon-related upstream regulators and functions related to calcium uptake and immune signaling. Several compounds in the targeted drug screen increased C7 expression in at least one keratinocyte line, but only kaempferol, a plant flavonoid, significantly increased C7 mRNA and protein in a DEB patient line. Conclusions: The novel C7_tdTomato reporter cell line can be used to screen compounds for increased C7 expression. The CRISPRa screen combined with a fluorescent reporter cell line can be used to reveal mechanistic regulators of gene expression and therapeutic targets for rare genetic diseases. Kaempferol has shown promising results in increasing C7 production and should be further evaluated as a potential therapeutic for DEB patients.
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