Protein overexpression: Mammalian stable cell line development

Hybridoma mammalian cell lines are hybrid cells produced from the fusion of normal cells with myeloma tumor cells. These cells constitutively produce specific antibodies from the primary lymphocytes and have characteristic immortalization. As a result, these cells continuously overexpress target proteins (including monoclonal antibodies) and do not go into senescence. Mammalian cells offer advantages in post-translational modification and protein folding that are crucial in the production of antibodies, recombinant proteins, viral-subunit proteins, and vectors for gene therapy. Unlike transient transfection, which provides short-term expression, stable cell lines integrate the gene of interest into the host genome, enabling continuous and controlled protein production. This capability is particularly beneficial for large-scale bioproduction of monoclonal antibodies, recombinant proteins, and viral vectors. However, the process of developing stable cell lines is complex, requiring careful selection of expression vectors, host cells, and screening methods to achieve optimal protein yield and stability. Protein overexpression in mammalian cells is a critical tool for research, drug discovery, and biopharmaceutical production. Stable cell line development ensures sustained and high-yield protein expression, which is essential for structural and functional studies, recombinant protein production, and therapeutic applications. This article discusses the key methodologies, challenges, and optimization strategies in developing mammalian stable cell lines for protein overexpression.

The choice of an expression vector is crucial for successful protein overexpression. Vectors typically contain a strong promoter (e.g., CMV, EF1α, or SV40), a selection marker (e.g., neomycin, puromycin, hygromycin), and regulatory elements to enhance transcription and translation efficiency. Codon optimization and the inclusion of signal peptides can further improve expression levels and protein secretion. Commonly used mammalian host cell lines for stable expression include Chinese hamster ovary (CHO) cells and mouse myeloma NS0 cells. CHO cells are widely used for industrial protein production due to their robust growth in suspension culture and high adaptability to bioreactor conditions.

Stable cell line development relies on integrating the gene of interest into the host genome. This can be achieved through traditional methods that involve transfection with plasmid DNA and antibiotic selection. While simple, this approach often results in variable expression due to position effects and epigenetic silencing. The site-specific integration with CRISPR/Cas9-mediated genome editing and recombinase-based systems (e.g., Flp-In, PiggyBac, or PhiC31) that enable targeted gene insertion, leading to more consistent and predictable protein expression. The dihydrofolate reductase (DHFR) and glutamine synthetase (GS) selection systems enable gene amplification, thereby increasing protein yield in CHO and NS0 cells. Following gene integration, selection and screening of high-expressing clones are essential. Limiting dilution cloning, fluorescence-activated cell sorting (FACS), and automated single-cell isolation methods help identify stable clones. High-throughput screening using enzyme-linked immunosorbent assay (ELISA), Western blotting, and flow cytometry ensures the selection of cell lines with optimal expression levels.

Long-term stability of protein expression is assessed through multiple passages under selective pressure. Once a stable clone is identified, process optimization for large-scale production involves adapting cells to serum-free and chemically defined media, optimizing bioreactor conditions (pH, temperature, oxygen supply), and monitoring product quality using analytical techniques such as mass spectrometry and glycan profiling. Despite advancements in stable cell line development, several challenges remain. Gene silencing, low integration efficiency, and clonal variability can hinder reproducibility and yield.

Hybridomas (antibody-producing cell lines) and stable cell lines overexpressing specific proteins are costly and vital assets for life science research. The generation of such protein-expressing cell lines can be a challenge requiring extensive cell culture experience and expertise; however, the production of such cell lines is very important to gene therapy, new compound screening, and cancer treatment research.

Stable cell lines have integrated plasmids in the genome that also produce gene specific overexpression of desired proteins (see generation of stable cell lines for more details). Our scientists bring years of extensive cell biology experience, pre-clinical CRO research, in vivo toxicology studies, xenograft services, teratoma formation, gene expression analysis, liposome encapsulation, and generation of stable cell lines.

At Altogen Labs, we offer development of protein overexpression clonal stable cell lines based on client specifications, all in a timely and cost-effective manner. Isolation and characterization of cell lines ensures our cell lines ideal models for in vitro pharmacology and bioengineering research.

Standard Cell Line Services:

• Cell engineering / stable cell line development
• Construct cloning, and subcloning, pDNA amplification and purification
• Stable transfection, drug selection
• Identification of protein overexpressing clones, colony isolation
• Clonal cell line development
• Characterization of cell line
• Validation of mRNA and protein expression (using qRT-PCR and/or Western blot analysis)

Download Altogen Labs Stable Cell Line Development PowerPoint Presentation: [PPT]

 

Altogen Labs is GLP-compliant research laboratory providing life science services, including variety of pre-clinical CRO studies, microorganism identification, bioremediation, RNAi gene silencing services: gene targeting, siRNA synthesis, chemical modification, functional in vitro validation, siRNA encapsulation, in vivo siRNA protection and tissue-targeted delivery. Altogen Labs is committed to offer highest quality service at the lowest price for scientific research for pharmaceutical and biotechnology industry and academic institutions.

Please let us know the details of your project and we will be happy to provide you an immediate price quote (contact e-mail: info@altogenlabs.com or call Altogen Labs at 512-433-6177). Experimental details will help us to provide an accurate quote and timeline estimate.

Get Instant Quote for
Generation of Stable Cell Line in 28 Days