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Updated: Aug 12, 2026
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Published on: March 22, 2016
Integration and stability of CHO amplicons containing plasmid sequences
F M Wurm1, M G Pallavicini, R Arathoon
1Genentech Inc., South San Francisco, CA 94080.
Developments in Biological Standardization
|January 1, 1992
Summary
Genetic stability in recombinant CHO cells was assessed using FISH and perfusion culture. Methotrexate (MTX) removal led to a decline in heterogeneous amplicons, favoring a master integration unit, maintaining productivity over 100 days.
Area of Science:
- Biotechnology
- Cell Biology
- Genetic Engineering
Background:
- Recombinant Chinese hamster ovary (CHO) cells are crucial for therapeutic protein production.
- Understanding genetic stability and amplicon structure is vital for consistent protein yield.
- Methotrexate (MTX) is commonly used for gene amplification and selection in CHO cells.
Purpose of the Study:
- To investigate the genetic stability and amplicon dynamics in recombinant CHO cells under varying culture conditions.
- To analyze the impact of MTX withdrawal on amplicon structure and frequency.
- To assess the long-term productivity and genetic integrity of CHO cells during non-selective perfusion culture.
Main Methods:
- Fluorescence in situ hybridization (FISH) was employed to visualize and analyze the distribution and structure of recombinant amplicons.
- High-density, non-selective long-term perfusion culture was utilized to mimic industrial production conditions.
- Recombinant CHO cell populations, including heterogeneous and cloned lines, were cultured with and without MTX.
Main Results:
- In the presence of MTX, 40-60% of cells exhibited multiple and/or unusually structured amplified regions.
- MTX removal rapidly decreased the frequency of heterogeneous amplified sequences, favoring a stable "master integration" unit.
- Long-term non-selective perfusion culture did not lead to a reduction in copy number or loss of amplified sequences, maintaining specific productivity over 100 days.
Conclusions:
- The "master integration" unit confers genetic stability in CHO cells upon MTX withdrawal.
- Non-selective perfusion culture maintains the genetic stability and high productivity of recombinant CHO cells.
- These findings support the use of non-selective conditions for long-term production of recombinant proteins from CHO cells.
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