Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants
Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (<i>LDLR</i>) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in...
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/14/11435 |
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| author | Jacek Jasiecki Monika Targońska Anna Janaszak-Jasiecka Magdalena Chmara Monika Żuk Leszek Kalinowski Krzysztof Waleron Bartosz Wasąg |
| author_facet | Jacek Jasiecki Monika Targońska Anna Janaszak-Jasiecka Magdalena Chmara Monika Żuk Leszek Kalinowski Krzysztof Waleron Bartosz Wasąg |
| author_sort | Jacek Jasiecki |
| collection | DOAJ |
| description | Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (<i>LDLR</i>) 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 <i>LDLR</i> 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–puromycin knock-in. The expressing vector with the <i>LDLR</i> 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 <i>LDLR</i> promoter–luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the <i>LDLR</i> 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 <i>LDLR</i> 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. |
| first_indexed | 2024-03-11T01:00:28Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T01:00:28Z |
| publishDate | 2023-07-01 |
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| spelling | doaj.art-390da28e41ce42919a922e64f522e3372023-11-18T19:39:24ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124141143510.3390/ijms241411435Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) VariantsJacek Jasiecki0Monika Targońska1Anna Janaszak-Jasiecka2Magdalena Chmara3Monika Żuk4Leszek Kalinowski5Krzysztof Waleron6Bartosz Wasąg7Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gdańsk, 80-416 Gdańsk, PolandDepartment of Biology and Medical Genetics, Medical University of Gdańsk, 80-210 Gdańsk, PolandDepartment of Medical Laboratory Diagnostics—Fahrenheit Biobank BBMRI.pl, Medical University of Gdańsk, 80-211 Gdańsk, PolandCenter of Translational Medicine, Medical University of Gdańsk, 80-210 Gdańsk, PolandDepartment of Biology and Medical Genetics, Medical University of Gdańsk, 80-210 Gdańsk, PolandDepartment of Medical Laboratory Diagnostics—Fahrenheit Biobank BBMRI.pl, Medical University of Gdańsk, 80-211 Gdańsk, PolandDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gdańsk, 80-416 Gdańsk, PolandDepartment of Biology and Medical Genetics, Medical University of Gdańsk, 80-210 Gdańsk, PolandFamilial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (<i>LDLR</i>) 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 <i>LDLR</i> 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–puromycin knock-in. The expressing vector with the <i>LDLR</i> 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 <i>LDLR</i> promoter–luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the <i>LDLR</i> 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 <i>LDLR</i> 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.https://www.mdpi.com/1422-0067/24/14/11435LDL receptorLDLRlow-density lipoproteinLDL uptakefamilial hypercholesterolemiaCRISPR |
| spellingShingle | Jacek Jasiecki Monika Targońska Anna Janaszak-Jasiecka Magdalena Chmara Monika Żuk Leszek Kalinowski Krzysztof Waleron Bartosz Wasąg Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants International Journal of Molecular Sciences LDL receptor LDLR low-density lipoprotein LDL uptake familial hypercholesterolemia CRISPR |
| title | Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants |
| title_full | Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants |
| title_fullStr | Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants |
| title_full_unstemmed | Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants |
| title_short | Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants |
| title_sort | novel tools for comprehensive functional analysis of ldlr low density lipoprotein receptor variants |
| topic | LDL receptor LDLR low-density lipoprotein LDL uptake familial hypercholesterolemia CRISPR |
| url | https://www.mdpi.com/1422-0067/24/14/11435 |
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