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

Fatemeh Safari,Mazyar Barekati-Mowahed,Safar Farajnia,Abbas Behzad Behbahani,Habib Zarredar,Hesam Dehghani

doi:10.1186/s40659-020-00319-x

Abstract

BackgroundChinese 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.ResultsFindings 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.ConclusionThese 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.

Highlights

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commer‐ cial-scale production of biopharmaceutical proteins
Application of clustered regularly interspaced short palindromic repeats (CRISPR) homology independent targeted integration (HITI) mediated knockout strategy for targeting CASP‐7 To target the active site of caspase 7, we aligned the sequence of this protein with the human caspase 7 sequence
Statistical analysis using ANOVA test showed that the silencing of caspase 7 reduced the viability of CHO-KO cells in comparison with wild type CHO-K1. (p-value: day 1 = 0.0001, day 2 = 0.0089, day 3 = 0.0138, day 5 = 0.0003). Analysis of these findings showed that the reduction of CHO-KO cell viability, in addition to Sodium butyrate (NaBu) cytotoxicity may be due to its lower cell proliferation

Summary

Introduction

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commer‐ cial-scale production of biopharmaceutical proteins. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. 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. Chinese hamster ovary (CHO) cells are the most commonly used cells for stable gene expression and producing heterologous proteins [1]. C The schematic of PX460-1 vector which contains a single cloning site for bait gRNA and the expression cassette of the EGFP gene in high density leads to environmental perturbations and cell stress due to the limitation of nutrients and oxygen, and accumulation of toxic metabolites [5] If the target site remains intact or with integration in the reverse direction, DNA bears additional cleavage since forward gene insertion or gRNA can no longer bind to the target site through errors from the NHEJ repair system. c The schematic of PX460-1 vector which contains a single cloning site for bait gRNA and the expression cassette of the EGFP gene in high density leads to environmental perturbations and cell stress due to the limitation of nutrients and oxygen, and accumulation of toxic metabolites [5]

Methods

Results

Discussion

Conclusion