{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T16:56:17Z","timestamp":1776012977302,"version":"3.50.1"},"reference-count":43,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,4,4]],"date-time":"2023-04-04T00:00:00Z","timestamp":1680566400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"U.S. National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AG066928"],"award-info":[{"award-number":["R01 AG066928"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving &gt;80\u201390% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development, and detailed characterization of a dual-function \u201cTRE-Lox\u201d system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of a target gene\u2014the lysosomal aspartyl protease cathepsin D (CatD)\u2014based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5\u2032 end of the endogenous CatD gene (CTSD). Using an optimized reverse-tetracycline transrepressor (rtTR) variant fused with the Kr\u00fcppel-associated box (KRAB) domain, we show that CatD expression can be disrupted by as much as 98% in mouse embryonic fibroblasts (MEFs). This system is highly sensitive to Dox (IC50 = 1.46 ng\/mL) and results in rapid (t1\/2 = 0.57 d) and titratable downregulation of CatD. Notably, even near-total disruption of CatD expression was completely reversed by withdrawal of Dox. As expected, transient expression of Cre recombinase results in complete deletion of the CTSD gene. The dual functionality of this novel system will facilitate future studies of the involvement of CatD in various diseases, particularly those attributable to partial loss of CatD function. In addition, the TRE-Lox approach should be applicable to the regulation of other target genes requiring more complete disruption than can be achieved by traditional methods.<\/jats:p>","DOI":"10.3390\/ijms24076745","type":"journal-article","created":{"date-parts":[[2023,4,5]],"date-time":"2023-04-05T02:11:41Z","timestamp":1680660701000},"page":"6745","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Dual-Function \u201cTRE-Lox\u201d System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D"],"prefix":"10.3390","volume":"24","author":[{"given":"Heather M.","family":"Terron","sequence":"first","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Derek S.","family":"Maranan","sequence":"additional","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"},{"name":"Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luke A.","family":"Burgard","sequence":"additional","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"},{"name":"Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frank M.","family":"LaFerla","sequence":"additional","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"},{"name":"Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shelley","family":"Lane","sequence":"additional","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7185-4255","authenticated-orcid":false,"given":"Malcolm A.","family":"Leissring","sequence":"additional","affiliation":[{"name":"Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA 92697, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6861","DOI":"10.1073\/pnas.89.15.6861","article-title":"Tissue- and site-specific DNA recombination in transgenic mice","volume":"89","author":"Orban","year":"1992","journal-title":"Proc. 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