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Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out

Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low...

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Autores principales: Zhang, Hao, Zhu, Shujuan, Xing, Yu, Liu, Qian, Guo, Zhen, Cai, Ziling, Shen, Zihao, Xia, Qingqian, Sheng, Huajun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8896708/
https://www.ncbi.nlm.nih.gov/pubmed/35245307
http://dx.doi.org/10.1371/journal.pone.0263162
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author Zhang, Hao
Zhu, Shujuan
Xing, Yu
Liu, Qian
Guo, Zhen
Cai, Ziling
Shen, Zihao
Xia, Qingqian
Sheng, Huajun
author_facet Zhang, Hao
Zhu, Shujuan
Xing, Yu
Liu, Qian
Guo, Zhen
Cai, Ziling
Shen, Zihao
Xia, Qingqian
Sheng, Huajun
author_sort Zhang, Hao
collection PubMed
description Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.
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spelling pubmed-88967082022-03-05 Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out Zhang, Hao Zhu, Shujuan Xing, Yu Liu, Qian Guo, Zhen Cai, Ziling Shen, Zihao Xia, Qingqian Sheng, Huajun PLoS One Research Article Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors. Public Library of Science 2022-03-04 /pmc/articles/PMC8896708/ /pubmed/35245307 http://dx.doi.org/10.1371/journal.pone.0263162 Text en © 2022 Zhang et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Zhang, Hao
Zhu, Shujuan
Xing, Yu
Liu, Qian
Guo, Zhen
Cai, Ziling
Shen, Zihao
Xia, Qingqian
Sheng, Huajun
Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title_full Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title_fullStr Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title_full_unstemmed Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title_short Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out
title_sort effect of cryopreservation on a172 and u251 glioma cells infected with lentiviral vectors designed for crispr/cas9-mediated aquaporin-8 knock-out
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8896708/
https://www.ncbi.nlm.nih.gov/pubmed/35245307
http://dx.doi.org/10.1371/journal.pone.0263162
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