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Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research
BACKGROUND: Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318917/ https://www.ncbi.nlm.nih.gov/pubmed/30606176 http://dx.doi.org/10.1186/s12896-018-0491-5 |
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author | Eun, Kiyoung Park, Min Gi Jeong, Yeon Woo Jeong, Yeon Ik Hyun, Sang-Hwan Hwang, Woo Suk Kim, Sung-Hak Kim, Hyunggee |
author_facet | Eun, Kiyoung Park, Min Gi Jeong, Yeon Woo Jeong, Yeon Ik Hyun, Sang-Hwan Hwang, Woo Suk Kim, Sung-Hak Kim, Hyunggee |
author_sort | Eun, Kiyoung |
collection | PubMed |
description | BACKGROUND: Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research. RESULTS: We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique. CONCLUSIONS: Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-018-0491-5) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6318917 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-63189172019-01-08 Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research Eun, Kiyoung Park, Min Gi Jeong, Yeon Woo Jeong, Yeon Ik Hyun, Sang-Hwan Hwang, Woo Suk Kim, Sung-Hak Kim, Hyunggee BMC Biotechnol Research Article BACKGROUND: Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research. RESULTS: We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique. CONCLUSIONS: Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-018-0491-5) contains supplementary material, which is available to authorized users. BioMed Central 2019-01-03 /pmc/articles/PMC6318917/ /pubmed/30606176 http://dx.doi.org/10.1186/s12896-018-0491-5 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Eun, Kiyoung Park, Min Gi Jeong, Yeon Woo Jeong, Yeon Ik Hyun, Sang-Hwan Hwang, Woo Suk Kim, Sung-Hak Kim, Hyunggee Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title | Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title_full | Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title_fullStr | Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title_full_unstemmed | Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title_short | Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research |
title_sort | establishment of tp53-knockout canine cells using optimized cripsr/cas9 vector system for canine cancer research |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318917/ https://www.ncbi.nlm.nih.gov/pubmed/30606176 http://dx.doi.org/10.1186/s12896-018-0491-5 |
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