Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

Abstract Background: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and...

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Autores principales:	Safari,Fatemeh, Farajnia,Safar, Behbahani,Abbas Behzad, Zarredar,Habib, Barekati-Mowahed,Mazyar, Dehghani,Hesam
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2020
Materias:	CHO cells Apoptosis CRISPR-associated protein 9 Caspase 7 Cell proliferatio
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602020000100239
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spelling	oai:scielo:S0716-976020200001002392020-11-24Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viabilitySafari,FatemehFarajnia,SafarBehbahani,Abbas BehzadZarredar,HabibBarekati-Mowahed,MazyarDehghani,Hesam CHO cells Apoptosis CRISPR-associated protein 9 Caspase 7 Cell proliferatio Abstract Background: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. Results: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. Conclusion: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.info:eu-repo/semantics/openAccessSociedad de Biología de ChileBiological Research v.53 20202020-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602020000100239en10.1186/s40659-020-00319-x
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	CHO cells Apoptosis CRISPR-associated protein 9 Caspase 7 Cell proliferatio
spellingShingle	CHO cells Apoptosis CRISPR-associated protein 9 Caspase 7 Cell proliferatio Safari,Fatemeh Farajnia,Safar Behbahani,Abbas Behzad Zarredar,Habib Barekati-Mowahed,Mazyar Dehghani,Hesam Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
description	Abstract Background: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. Results: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. Conclusion: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
author	Safari,Fatemeh Farajnia,Safar Behbahani,Abbas Behzad Zarredar,Habib Barekati-Mowahed,Mazyar Dehghani,Hesam
author_facet	Safari,Fatemeh Farajnia,Safar Behbahani,Abbas Behzad Zarredar,Habib Barekati-Mowahed,Mazyar Dehghani,Hesam
author_sort	Safari,Fatemeh
title	Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
title_short	Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
title_full	Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
title_fullStr	Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
title_full_unstemmed	Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability
title_sort	caspase-7 deficiency in chinese hamster ovary cells reduces cell proliferation and viability
publisher	Sociedad de Biología de Chile
publishDate	2020
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602020000100239
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Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

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