Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters
ABSTRACT The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitatio...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-01-01
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| Series: | JBMR Plus |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jbm4.10706 |
| _version_ | 1797948054368681984 |
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| author | Wenhuan Chai Weiwei Hao Jintao Liu Zhenglin Han Shiyu Chang Liben Cheng Mingxin Sun Guofang Yan Zemin Liu Yin Liu Guodong Zhang Li Xing Hongqian Chen Peng Liu |
| author_facet | Wenhuan Chai Weiwei Hao Jintao Liu Zhenglin Han Shiyu Chang Liben Cheng Mingxin Sun Guofang Yan Zemin Liu Yin Liu Guodong Zhang Li Xing Hongqian Chen Peng Liu |
| author_sort | Wenhuan Chai |
| collection | DOAJ |
| description | ABSTRACT The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so‐called nonspecific/off‐target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk‐Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk‐Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk‐Cre expression at the single‐cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT‐nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT‐PCR. The results demonstrate that Ctsk‐Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk‐Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk‐Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. |
| first_indexed | 2024-04-10T21:37:34Z |
| format | Article |
| id | doaj.art-efbe7966d4e84de4afc1f8150ed22f77 |
| institution | Directory Open Access Journal |
| issn | 2473-4039 |
| language | English |
| last_indexed | 2024-04-10T21:37:34Z |
| publishDate | 2023-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | JBMR Plus |
| spelling | doaj.art-efbe7966d4e84de4afc1f8150ed22f772023-01-19T11:32:55ZengWileyJBMR Plus2473-40392023-01-0171n/an/a10.1002/jbm4.10706Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP ReportersWenhuan Chai0Weiwei Hao1Jintao Liu2Zhenglin Han3Shiyu Chang4Liben Cheng5Mingxin Sun6Guofang Yan7Zemin Liu8Yin Liu9Guodong Zhang10Li Xing11Hongqian Chen12Peng Liu13Laboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaLaboratory of Bone & Adipose Biology Shanxi Medical University Taiyuan ChinaABSTRACT The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so‐called nonspecific/off‐target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk‐Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk‐Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk‐Cre expression at the single‐cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT‐nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT‐PCR. The results demonstrate that Ctsk‐Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk‐Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk‐Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.https://doi.org/10.1002/jbm4.10706AGINGCATHEPSIN KCREGFPSOSTEOBLASTSOSTEOCLASTS |
| spellingShingle | Wenhuan Chai Weiwei Hao Jintao Liu Zhenglin Han Shiyu Chang Liben Cheng Mingxin Sun Guofang Yan Zemin Liu Yin Liu Guodong Zhang Li Xing Hongqian Chen Peng Liu Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters JBMR Plus AGING CATHEPSIN K CRE GFPS OSTEOBLASTS OSTEOCLASTS |
| title | Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters |
| title_full | Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters |
| title_fullStr | Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters |
| title_full_unstemmed | Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters |
| title_short | Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters |
| title_sort | visualizing cathepsin k cre expression at the single cell level with gfp reporters |
| topic | AGING CATHEPSIN K CRE GFPS OSTEOBLASTS OSTEOCLASTS |
| url | https://doi.org/10.1002/jbm4.10706 |
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