Abstract: PO0386
CRISPR-Induced Knockout of Ubiquitin Ligase Cullin 3 in Human Proximal Tubule Cells
Session Information
- AKI: Mechanisms of Injury
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Saritas, Turgay, University Hospital RWTH Aachen, Aachen, Germany
- Chen, Lu, University Hospital RWTH Aachen, Aachen, Germany
- Ziegler, Susanne, University Hospital RWTH Aachen, Aachen, Germany
- Kabgani, Nazanin, University Hospital RWTH Aachen, Aachen, Germany
- Kuppe, Christoph, University Hospital RWTH Aachen, Aachen, Germany
- Floege, Jürgen, University Hospital RWTH Aachen, Aachen, Germany
- McCormick, James A., Oregon Health & Science University School of Medicine, Portland, Oregon, United States
- Kramann, Rafael, University Hospital RWTH Aachen, Aachen, Germany
Background
We have shown earlier that kidney-specific deletion of Cullin 3 (Cul3) causes proximal tubule injury and fibrosis, but the mechanism is still unclear. Cul3 is essential for the ubiquitination and thus degradation of many critical proteins in several organs. This study aims to generate a Cul3-deficient human proximal tubule cell line using the CRISPR-Cas9 system. This model will allow us to understand the mechanistic role of Cul3 in the human proximal tubule.
Methods
CD10+ proximal tubule cells were isolated by cell sorting from healthy human kidney cortex of a nephrectomy specimen. The primary proximal tubule cells were cultivated and then immortalized using SV40LT and HTERT. Using different CRISPR/Cas9 approaches, Cul3 knockout clones were aimed to achieve. The most successful approach is depicted in the results section.
Results
The Cul3-specific guide RNA was cloned into pL-CRISPR.EFS.GFP using BsmBI restriction digestion. Lentiviral particles were produced by transient co-transfection of HEK293T cells with lentiviral transfer plasmid, packaging plasmid psPAX2 and VSVG packaging plasmid pMD2.G using TransIT-LT. Viral supernatants were collected 48–72 h after transfection, clarified by centrifugation, supplemented with 10% FCS and polybrene and sterile filtered. Cell transduction was performed by incubating the CD10+ cells with viral supernatants. eGFP-expressing cells were single-cell sorted into 96-well plates. Expanded colonies were assessed for mutations with mismatch detection assay: gDNA spanning the CRISPR target site was PCR amplified and analyzed by T7EI digest. To determine specific mutation events on both alleles within the clones grown, the PCR product was subcloned into the pCR 4Blunt-TOPO vector. Minimum 6 colonies per CRISPR-clone were grown and sent for sanger sequencing. Results from qPCR and western blot confirmed Cul3 knockout. The clones were sent out for bulk RNA sequencing to reveal differentially regulated genes upon Cul3 deletion.
Conclusion
Cul3 is a major upstream player in different cell signaling pathways. This study will show the role of Cul3 in the proximal tubule of human kidney.
Funding
- Government Support – Non-U.S.