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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.

ABSTRACT: Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of ?-galactosidase A (?-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable ?-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of ?-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.

SUBMITTER: Pereira EM

PROVIDER: S-EPMC5130460 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.

Pereira Ester M EM Labilloy Anatália A Eshbach Megan L ML Roy Ankita A Subramanya Arohan R AR Monte Semiramis S Labilloy Guillaume G Weisz Ora A OA

American journal of physiology. Renal physiology 20160928 5

Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized ...[more]

PMID: 27681560

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Project description:The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.

Dataset Information

Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.

Publications

Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.

Similar Datasets

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