2. Academic Validation
  3. Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

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

  • Biol Res. 2020 Nov 13;53(1):52. doi: 10.1186/s40659-020-00319-x.
Fatemeh Safari 1 2,  Safar Farajnia 3 4,  Abbas Behzad Behbahani 2,  Habib Zarredar 5,  Mazyar Barekati-Mowahed 6,  Hesam Dehghani 7
Affiliations

Affiliations

  • 1 Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
  • 2 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
  • 3 Biotechnology Research Center, Tabriz University of Medical Sciences, Daneshgah Ave., Tabriz, Iran. [email protected].
  • 4 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. [email protected].
  • 5 Tuberculosis and Lung Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
  • 6 Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
  • 7 Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
PMID: 33187557 DOI: 10.1186/s40659-020-00319-x
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.

Keywords

Apoptosis; CHO cells; CRISPR-associated protein 9; Caspase 7; Cell proliferatio.

Figures