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Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2
mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopress...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866188/ https://www.ncbi.nlm.nih.gov/pubmed/36675199 http://dx.doi.org/10.3390/ijms24021684 |
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author | Jang, Hyo-Ju Park, Hye-Jeong Choi, Hong Seok Jung, Hyun Jun Kwon, Tae-Hwan |
author_facet | Jang, Hyo-Ju Park, Hye-Jeong Choi, Hong Seok Jung, Hyun Jun Kwon, Tae-Hwan |
author_sort | Jang, Hyo-Ju |
collection | PubMed |
description | mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3′- and 5′-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies. |
format | Online Article Text |
id | pubmed-9866188 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-98661882023-01-22 Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 Jang, Hyo-Ju Park, Hye-Jeong Choi, Hong Seok Jung, Hyun Jun Kwon, Tae-Hwan Int J Mol Sci Article mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3′- and 5′-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies. MDPI 2023-01-14 /pmc/articles/PMC9866188/ /pubmed/36675199 http://dx.doi.org/10.3390/ijms24021684 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Jang, Hyo-Ju Park, Hye-Jeong Choi, Hong Seok Jung, Hyun Jun Kwon, Tae-Hwan Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title | Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title_full | Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title_fullStr | Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title_full_unstemmed | Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title_short | Genome-Engineered mpkCCDc14 Cells as a New Resource for Studying AQP2 |
title_sort | genome-engineered mpkccdc14 cells as a new resource for studying aqp2 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9866188/ https://www.ncbi.nlm.nih.gov/pubmed/36675199 http://dx.doi.org/10.3390/ijms24021684 |
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