Recombinant protein expression by targeting pre-selected chromosomal loci.

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Recombinant protein expression in mammalian cells relies on transgene integration into chromosomal DNA.
• Chromosomal location significantly influences transgene expression levels.
• Targeting specific loci with defined regulatory elements can optimize expression systems.

Purpose of the Study:

• To explore antibody expression by targeting diverse constructs into pre-tagged chromosomal loci in CHO-K1 and HEK293 cells.
• To evaluate the impact of chromosomal surroundings on distinct vector constructs.
• To demonstrate the efficacy of Flp recombinase mediated cassette exchange (RMCE) for predictable protein expression.

Main Methods:

• Cells (CHO-K1, HEK293) were tagged with reporter gene expression cassettes via retroviral infection or plasmid transfection.
• Tagged cell clones with single-copy integration and high reporter expression were selected.
• Flp recombinase mediated cassette exchange (RMCE) was used to target antibody expression cassettes into selected, FRT-flanked loci.

Main Results:

• Cell clones producing > 20 pg/cell/24 hours were identified.
• RMCE achieved 90-100% correct integration of antibody expression cassettes.
• Antibody production was highly consistent within isogenic cell clones.
• Promoter strength was critically dependent on the chromosomal context, demonstrating its dominant influence.

Conclusions:

• RMCE is a powerful and accurate method for designing optimized gene expression vectors.
• This approach allows for the exploitation of specific chromosomal sites for predictable expression of biotechnologically relevant proteins like antibodies.