{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T17:57:16Z","timestamp":1791482236317,"version":"4.3.4"},"reference-count":35,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2023,7,14]],"date-time":"2023-07-14T00:00:00Z","timestamp":1689292800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"NCN","doi-asserted-by":"publisher","award":["OPUS 2015\/19\/B\/NZ5\/03510"],"award-info":[{"award-number":["OPUS 2015\/19\/B\/NZ5\/03510"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004281","name":"NCN","doi-asserted-by":"publisher","award":["10\/E-389\/SPUB\/SP\/2020"],"award-info":[{"award-number":["10\/E-389\/SPUB\/SP\/2020"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Ministry of Education and Science Poland","award":["OPUS 2015\/19\/B\/NZ5\/03510"],"award-info":[{"award-number":["OPUS 2015\/19\/B\/NZ5\/03510"]}]},{"name":"Ministry of Education and Science Poland","award":["10\/E-389\/SPUB\/SP\/2020"],"award-info":[{"award-number":["10\/E-389\/SPUB\/SP\/2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR\/Cas9-mediated luciferase\u2013puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter\u2013luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.<\/jats:p>","DOI":"10.3390\/ijms241411435","type":"journal-article","created":{"date-parts":[[2023,7,14]],"date-time":"2023-07-14T08:35:47Z","timestamp":1689323747000},"page":"11435","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8192-6486","authenticated-orcid":false,"given":"Jacek","family":"Jasiecki","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gda\u0144sk, 80-416 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5613-189X","authenticated-orcid":false,"given":"Monika","family":"Targo\u0144ska","sequence":"additional","affiliation":[{"name":"Department of Biology and Medical Genetics, Medical University of Gda\u0144sk, 80-210 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2695-4756","authenticated-orcid":false,"given":"Anna","family":"Janaszak-Jasiecka","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Diagnostics\u2014Fahrenheit Biobank BBMRI.pl, Medical University of Gda\u0144sk, 80-211 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena","family":"Chmara","sequence":"additional","affiliation":[{"name":"Center of Translational Medicine, Medical University of Gda\u0144sk, 80-210 Gda\u0144sk, Poland"},{"name":"Laboratory of Clinical Genetics, University Clinical Centre, 80-952 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"\u017buk","sequence":"additional","affiliation":[{"name":"Department of Biology and Medical Genetics, Medical University of Gda\u0144sk, 80-210 Gda\u0144sk, Poland"},{"name":"Laboratory of Clinical Genetics, University Clinical Centre, 80-952 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7270-1592","authenticated-orcid":false,"given":"Leszek","family":"Kalinowski","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Diagnostics\u2014Fahrenheit Biobank BBMRI.pl, Medical University of Gda\u0144sk, 80-211 Gda\u0144sk, Poland"},{"name":"BioTechMed Centre, Department of Mechanics of Materials and Structures, Gdansk University of Technology, 80-233 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9989-0984","authenticated-orcid":false,"given":"Krzysztof","family":"Waleron","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gda\u0144sk, 80-416 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3634-7562","authenticated-orcid":false,"given":"Bartosz","family":"Was\u0105g","sequence":"additional","affiliation":[{"name":"Department of Biology and Medical Genetics, Medical University of Gda\u0144sk, 80-210 Gda\u0144sk, Poland"},{"name":"Laboratory of Clinical Genetics, University Clinical Centre, 80-952 Gda\u0144sk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,14]]},"reference":[{"key":"ref_1","unstructured":"Valle, D.L., Antonarakis, S., Ballabio, A., Beaudet, A.L., and Mitchell, G.A. 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