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Renin Gene Editing in Zebrafish
Author(s) -
Mullins Linda Jane,
Hoffmann Scott,
Rider Sebastien,
Bradley Mark,
Mullins John
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.lb535
Subject(s) - zebrafish , biology , pronephros , microbiology and biotechnology , kidney development , knockout mouse , phenotype , renin–angiotensin system , kidney , gene knockout , gene , embryonic stem cell , genetics , endocrinology , blood pressure
Recent studies in zebrafish show that the renin‐angiotensin system (RAS) is highly conserved across vertebrates. In common with mammals, the zebrafish kidney contains specialised renin‐expressing mural cells throughout the kidney micro‐vasculature that contain acidic granules indicative of the processing and regulated secretion of active renin. Zebrafish are a unique model organism suitable for developmental studies and due to the translucent nature of zebrafish larvae, the functional embryonic pronephros is readily accessible for high‐resolution in vivo imaging and high‐throughput genetic and chemical screens. To investigate the role of renin and the RAS in zebrafish we designed guide RNAs to target CRISPR‐Cas9 to renin exon 2. We identified an 8bp deletion resulting in a frameshift mutation and early termination of renin transcription. Adult ren −/− knockouts, identified by sequencing, were phenotypically screened using the automated screening system (VAST), which permits the imaging of large numbers of live fish with reproducible precision. Knockout fish are viable but exhibit delayed growth during early stages of development. Imaging revealed a delayed appearance and enlargement of the swim bladder and reduced fish length was indicative of an overall developmental delay. The knockout fish were further crossed with already existing renal reporter lines to investigate further phenotypes at a cellular level. When ren −/− knockout fish were crossed with wt1b:GFP fish a delay in glomerular fusion of the pronephric kidney was observed, whilst a cross of ren −/− knockout with ren:RFP fish revealed a dramatic increase in renin cell number along the renal vasculature, suggesting a continued but non‐critical requirement for renin in the adult fish. Support or Funding Information BHF Centre of Research Excellence Award EPSRC/MRC CDT (Optima) Award This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .