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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

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Evaluating Apoptotic Gene Efficiency for CHO Culture Performance Using Targeted Integration.

David Catalán-Tatjer¹, Saravana Kumar Ganesan¹, Iván Martínez-Monje¹

¹The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby 2800, Denmark.

ACS Synthetic Biology

|April 23, 2025

Summary

Overexpressing Chinese hamster ovary (CHO) bcl-2 significantly improved biopharmaceutical production. This study established a targeted gene integration method to overcome challenges in metabolic engineering for cell culture performance.

Keywords:

CHO cells antiapoptosis targeted integration

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Area of Science:

Biotechnology
Cell Biology
Bioprocessing

Background:

Chinese hamster ovary (CHO) cells are crucial for biopharmaceutical production, but cell death limits culture performance.
Apoptosis (programmed cell death) is a key factor affecting CHO cell culture duration and density.
Previous studies faced challenges due to clonal variation from random gene integration.

Purpose of the Study:

To develop isogenic CHO cell lines for studying antiapoptotic genes.
To identify specific antiapoptotic genes that enhance biopharmaceutical production.
To establish a reliable method for testing candidate genes in CHO cell engineering.

Main Methods:

Utilized recombinase-mediated cassette exchange to create isogenic CHO cell lines expressing erythropoietin and various antiapoptotic genes (bcl-2, bcl-xL, mcl-1, bhrf-1).
Tested cell lines in batch cultures with sodium butyrate and in fed-batch using the ambr15 microbioreactor system.
Employed multiplexed quantitative proteomics to characterize metabolic differences.

Main Results:

Overexpression of CHO-origin bcl-2 significantly enhanced productivity.
Phenotypic variations were observed depending on the specific antiapoptotic gene overexpressed.
A methodology for targeted gene testing was successfully established.

Conclusions:

Targeted integration of antiapoptotic genes, specifically CHO bcl-2, can improve CHO cell culture productivity.
The developed methodology enables more comparable metabolic engineering strategies.
This research overcomes previous challenges associated with clonal variation in CHO cell line development.