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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
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PEI-Mediated Transient Gene Expression in CHO Cells.

Yashas Rajendra1

  • 1Denali Therapeutics, South San Francisco, CA, USA. rajendra@dnli.com.

Methods in Molecular Biology (Clifton, N.J.)
|June 26, 2024
PubMed
Summary
This summary is machine-generated.

This study presents a scalable polyethyleneimine (PEI)-mediated transient transfection method for Chinese hamster ovary (CHO-DG44) cells, optimizing protein expression from 2 mL to 2 L. The protocol involves high cell density transfection and specific incubation conditions for efficient gene delivery.

Keywords:
CHO cellsOrbital shakingPolyethyleneimineRecombinant proteinTransfection

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Molecular Biology

Background:

  • Transient transfection is crucial for recombinant protein production in mammalian cells.
  • Optimizing transfection efficiency and scalability remains a key challenge for industrial applications.
  • Chinese Hamster Ovary (CHO) cells are widely used for biopharmaceutical manufacturing.

Purpose of the Study:

  • To develop and describe a robust, scalable method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted CHO-DG44 cells.
  • To optimize conditions for high-level protein expression across a wide range of volumes (2 mL to 2 L).
  • To provide detailed protocols for successful gene delivery and protein production.

Main Methods:

  • Transfection of CHO-DG44 cells at high density (5 × 10^6 cells/mL) using direct addition of plasmid DNA (pDNA) and PEI.
  • Incubation at 31°C with orbital shaking for optimal transfection and protein production.
  • Utilized specific concentrations of coding pDNA, filler DNA, and PEI, with optional N,N-dimethylacetamide (DMA) addition.

Main Results:

  • Successfully demonstrated a scalable transfection method applicable from small-scale (2 mL) to large-scale (2 L) cultures.
  • Identified optimal reagent concentrations: 0.3 mg/L coding pDNA, 2.7 mg/L filler DNA, and 15 mg/L PEI.
  • Showcased a modified protocol to eliminate DMA by adjusting coding DNA levels, offering flexibility in production.

Conclusions:

  • The described PEI-mediated transfection method offers a reliable and scalable approach for protein expression in CHO-DG44 cells.
  • The protocol is adaptable for various culture volumes, facilitating process development and manufacturing.
  • This method provides a valuable tool for efficient recombinant protein production in biopharmaceutical research and industry.