Project description:We generated NUP133 WT and KO podocytes to model human hereditary SRNS in vitro. A subtype of SRNS is caused by monogenetic mutations of the nuclear pore complex including NUP133. Immortalized human podocytes were genome edited applying the CRISPR/Cas9 technique and two independent NUP133 sgRNAs. Two monoclonal cell backgrounds were used to create two matching sets of NUP133 WT and KO cells (WT-1 versus KO-1 and WT-2 versus KO-2). Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis was performed in duplicate for each cell line. NUP133 KO podocytes exhibit vast transcriptional changes compared to WT cells.
Project description:We generated EPB41L5 WT and KO podocytes to model human glomerular kidney disease in vitro. Immortalized human podocytes were genome edited applying the CRISPR/Cas9 technique. Two monoclonal cell lines were generated for WT and KO genotypes (WT-1, WT-2, KO-1 & KO-2). Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis was performed in duplicate for each cell line. EPB41L5 KO podocytes exhibit transcriptional changes compared to WT cells. Analyzed cell were previously described: Schell C, Rogg M, Suhm M, et al. The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier. Proc Natl Acad Sci U S A. 2017;114(23):E4621-E4630. doi:10.1073/pnas.1617004114
Project description:We generated NUP133 mutant podocytes to model human hereditary SRNS in vitro. A subtype of SRNS is caused by monogenetic mutations of the nuclear pore complex including NUP133. Human immortalized podocytes were genome edited applying the CRISPR/Cas9 technique to create NUP133 KO cells (“KO-1”). cDNA of NUP133-WT or causative variants NUP133 c.2922T>G (“Mutation-1”) or NUP133 c.3335-11T>A (“Mutation-2”) were stable expressed into this KO background by lentiviral transduction. Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis was performed in triplicate for each cell line. NUP133 mutant podocytes showed no overt transcriptional changes compared to NUP133-WT cells.
Project description:EGFP (control) and TEADi expressing podocytes were generated by lentivirus transduction to analyze transcriptional signaling by YAP/TAZ-TEAD. Tetracycline-inducible TEADi is a GFP-tagged inhibitor of the interaction of YAP1 and TAZ with TEAD transcription factors. pInducer20 EGFP-TEADi was a gift from Ramiro Iglesias-Bartolome (Addgene plasmid # 140145 ; http://n2t.net/addgene:140145 ; RRID:Addgene_140145). TEADi was previously described: YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity. Yuan Y., Park J., Feng A., Awasthi P., Wang Z., Chen Q., Iglesias-Bartolome R.. Nat Commun 11, 1472 (2020). 10.1038/s41467-020-15301-0. Transcriptome profiling (RNA-Sequencing) and differential gene expression analysis of 3 independent replicates per genotype was performed. TEADi podocytes exhibit transcriptional changes compared to WT cells including downregulation of prominent YAP target genes like CTGF, CYR61 and ANKRD1.
Project description:Purpose: The goal of this study is to identify the differential cardiac transcriptome profiling between WT and Smyd1 null (Smyd1-KO) hearts at E9.5 using RNA-seq. Methods: mRNA profiles of E9.5 WT and Smyd1-KO mouse hearts were generated by deep sequencing, n=3 for each genotype, using Illumina HiSeq2500. The sequence reads were aligned to the mm10 reference genome using STAR via the bcbio-nextgen RNA-sequencing pipeline. Differential gene expression was determined by DEseq2. Results: 1756 genes were differentially expressed between WT and Smyd1-KO hearts [adjusted P value <0.05, |log2(Fold Change)| > 0.5], with 1130 upregulated and 626 downregulated in E9.5 Smyd1-KO hearts.