Di and David will be presenting @ Student Seminar on 10.30.08
We will be having Student Seminar in 2289 CBRB @ noon on Thursday (10.30.08). For those of you that are interested in some of the related background, I’ve asked Di and David to provide something to whet your whistle. See you Thursday.
Genetic Disruption of Secreted Renin Preserves Expression of mRNA Encoding Intracellular Renin in the Brain
1Interdisciplinary Genetics Program, 2Department of Ob/Gyn, 3Department of Internal Medicine, and 4Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
Secreted renin (sRen) (NCBI Gene, OMIM, Wikipedia) is translated into a precursor encoding a signal peptide sorting preprorenin into renal JG cell vesicles where the prosegment is cleaved and the secretory pathway. We and others reported the discovery of a unique form of intracellular renin (icRen) in the brain of mouse, rat and human. icRen initiates transcription from an alternative promoter, utilizes a new 1st exon (exon-1b) and translates renin from a highly conserved ATG in exon-2. The translation product lacks: 1) the signal peptide (renin remains intracellular), and 2) part of the prosegment (prorenin is constitutively active). In the mouse genome exon-1b is located in the 1st intron of the gene thus providing an arrangement to delete sRen in kidney while preserving icRen in brain and vice versa. We performed gene targeting in C57BL/6 ES cells to ablate sRen while preserving icRen. We deleted sRen by “floxing” a region encoding exon-1a (including the canonical ATG) and extending approximately 500 bp upstream (deleting the classic promoter) and downstream (into intron A). The neo and floxed-regions were removed to generate floxed and null alleles. +/- mice carrying the null allele were intercrossed but only three -/- mice survived to weaning indicating lethality (+/+ 32; +/- 67; -/- 3). We are performing daily saline injection to newborns to rescue them. One -/- survivor demonstrated lower systolic blood pressure and less white adipose. We performed timed breedings between +/- mice to obtain -/- tissues at 18.5gd. Unlike the post-weanling mice, +/+, +/- and -/- fetuses were obtained in expected numbers (+/+ 7; +/- 20; -/- 9). RNA from fetal kidney and brain was examined with primers specific to sRen and icRen. Expression of sRen mRNA in kidney, and of sRen and icRen mRNA in brain was detected in +/- mice. However, -/- fetuses exhibited a loss of sRen in kidney and brain, but retained expression of icRen mRNA. We conclude: 1) deleting the coding potential for sRen does not affect production of icRen mRNA, 2) preservation of icRen in the brain is insufficient to rescue lethality caused by deletion of sRen, and 3) adult sRen systemic KO has lower blood pressure and significantly less white adipose. We are breeding the floxed allele to Nestin/GFAP-CRE mice to generate brain-specific KOs of sRen while reserving icRen. These novel models should provide data on the physiological relevance of intracellular renin in the brain.