Authors: Sidney Broome, Kyla Boyd, Maggie Holzworth, Zachary Levine, Lauren Douma, Michelle Gumz, and Kevin Brown
Faculty Mentor: Kevin Brown
College: College of Medicine
The peptide hormone Endothelin-1 (ET-1) acts to lower blood pressure through natriuretic events in the kidney’s renal collecting ducts. A recently discovered long, non-coding RNA, termed Edn1-AS, is transcribed in an antisense orientation to Edn1, the peptide’s encoding gene, and signifies a novel regulatory mechanism for ET-1 expression. Using CRISPR/Cas9 technology, the Gumz lab attempted to knock out an approximately 350 bp sequence of the Edn1-AS promoter in a mouse-derived inner medullary collecting duct (mIMCD-3) cell line. After obtaining a cell line with a monoallelic knockout, termed C8, our lab sought to delete the remaining wild-type allele by designing sgRNAs and cloning them into two CRISPR plasmids with different selectable markers. PCR analysis of one clone (C8-N) indicated a potential homozygous deletion. Sequencing of the Edn1-AS promoter within C8-N clone resulted in the detection of only the C8 deletion (Allele A). Using new primers recessed off the 5’ and 3’ termini of AlleleA indicated that Allele B contains an approximately 700 bp deletion that has a 26 bp overlap with the deleted region in allele A. Future studies are aimed at examining if ET-1 expression differs between parental mIMCD3, C8, and C8-N cells.