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CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CR...

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Autores principales: Sui, Chao, Jiang, Dandan, Wu, Xiangju, Cong, Xiaoyan, Li, Feng, Shang, Yingli, Wang, Jinqiu, Liu, Sidang, Shan, Hu, Qi, Jing, Du, Yijun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421005/
https://www.ncbi.nlm.nih.gov/pubmed/30941371
http://dx.doi.org/10.1155/2019/7398208
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author Sui, Chao
Jiang, Dandan
Wu, Xiangju
Cong, Xiaoyan
Li, Feng
Shang, Yingli
Wang, Jinqiu
Liu, Sidang
Shan, Hu
Qi, Jing
Du, Yijun
author_facet Sui, Chao
Jiang, Dandan
Wu, Xiangju
Cong, Xiaoyan
Li, Feng
Shang, Yingli
Wang, Jinqiu
Liu, Sidang
Shan, Hu
Qi, Jing
Du, Yijun
author_sort Sui, Chao
collection PubMed
description Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.
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spelling pubmed-64210052019-04-02 CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication Sui, Chao Jiang, Dandan Wu, Xiangju Cong, Xiaoyan Li, Feng Shang, Yingli Wang, Jinqiu Liu, Sidang Shan, Hu Qi, Jing Du, Yijun Biomed Res Int Research Article Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine. Hindawi 2019-03-03 /pmc/articles/PMC6421005/ /pubmed/30941371 http://dx.doi.org/10.1155/2019/7398208 Text en Copyright © 2019 Chao Sui et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Sui, Chao
Jiang, Dandan
Wu, Xiangju
Cong, Xiaoyan
Li, Feng
Shang, Yingli
Wang, Jinqiu
Liu, Sidang
Shan, Hu
Qi, Jing
Du, Yijun
CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title_full CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title_fullStr CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title_full_unstemmed CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title_short CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication
title_sort crispr-cas9 mediated rnase l knockout regulates cellular function of pk-15 cells and increases prv replication
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421005/
https://www.ncbi.nlm.nih.gov/pubmed/30941371
http://dx.doi.org/10.1155/2019/7398208
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