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: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: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: Next-generation sequencing (NGS) was used to define the transcriptome of native mouse podocytes and non-podocytes glomerular cells as part of a project aiming to define the molecular fingerprint of mouse podocytes. Method: Glomeruli from 29 Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J x hNPHS2Cre mice at the age of 10 weeks were purified and a single cell solution was prepared to seperate GFP-expressing (podocytes) and GFP-negative (non-podocytes glomerular cells) cells by FACS sorting. RNA was extracted and prepared for further analysis using directional, polyA+ library preparation. An Illumina HiSeq2500 was used for a paired-end sequencing of 100 cycles . Salmon and Sleuth were used for downstream analysis. Results: A total of 100 Million reads each from podocytes and non-podocytes glomerular cells could be used for further analysis.
Project description:Podocytes play an important filtration role in the kidney. We examined culture condition for efficient podocyte induction and established a method to selectively induce podocytes from human iPS cells. To understand how expression profiles of human iPS cell-derived podocytes were close to that in vivo, we isolated human adult podocytes for human adult kidney. Purified RNAs from human iPS cells, nephron progenitor cells, human immortalized podocyte cell line, human iPS cell-derived podocytes, and sorted human adult podocytes were analyzed by RNA-seq.
Project description:Transcriptome analysis of growth hormone dependant genes in glomerular podocytes Differentiated human glomerular podocytes in culture exposed to growth hormone for 0 min, 2 min, 5 min, 15 min, and 30 min. Total RNA is extracted and subjected to microarray analysis.