Mastering Mammalian Cell Transfection: A Step-by-Step Experiment Guide

The mammalian cell expression system has become the main recombinant protein production system for clinical applications, and the development of mammalian cell protein drugs has also become a trend in the research and development of biopharmaceutical products. In the past twenty years, significant progress has been made in research on mammalian expression vector construction, codon optimization, gene amplification methods, transfection methods, and other related areas. By designing appropriate mammalian expression vectors, mammalian cell expression can be improved, such as using strong promoters, appropriate signal peptides, codon optimization, etc. Gene amplification methods are often used to produce recombinant proteins on a large scale in mammalian cell expression systems, but due to significant differences between cells and unpredictable expression stability, screening is required to obtain high-yield proteins, which takes some time. Gene transfection can transfer exogenous nucleic acids, such as DNA, RNA, plasmids, etc., into eukaryotic cells while maintaining their biological functions. Through transfection, mammalian protein customization services can be provided. Cell transfection technology can be used to detect mammalian cell expression and can also produce recombinant proteins on a large scale for mammalian protein customization and mammalian cell protein drug development.

Methods and Classification of Mammalian Cell Transfection

In mammalian cell expression systems, the production of recombinant proteins is mainly achieved through transient transfection or stable transfection. Among them, the transient transfection operation is simple, and the exogenous nucleic acid does not integrate into the cell genome, but exists on the vector, which can express the target gene in a short period of time and is mostly used for small-scale protein synthesis in the short term. Stable transfection is the process of integrating exogenous nucleic acids into the cell genome, retaining them within the cell, and replicating them as part of the cell genome, cells can express target genes for a long time. And stable transfection can be used for long-term experimental research, large-scale protein production, and mammalian cell protein drug development.

The main methods of cell transfection include physical transfection (electroporation, microinjection, gene gun, etc.), chemical transfection (liposomes and their substitutes, calcium phosphate, etc.), and biological transfection (various viruses, including adenovirus, lentivirus, retrovirus, adenovirus, etc.). The common principle of these transfection methods is that a negatively charged nucleic acid binds to a positively charged compound to form a complex, which is then attracted to the negatively charged cell membrane and enters the cell through phagocytosis.

Tab 1. Transfection methods and their advantages

Conventional Liposome Transfection Method

The transfection efficiency mediated by liposomes is very high, and it can transfect some cells that are not easily transfected by other chemical methods. It can also mediate the transfer of nucleic acids into the human body, which can be used for gene therapy. Before carrying out liposome transfection, the cells should be seeded into a culture plate and cultured to an appropriate density, usually 70% -80%. Afterwards, the old culture medium should be discarded and washed twice with serum-free medium or PBS to remove residual serum and antibiotics and prevent them from affecting the transfection efficiency.

The commonly used transfection reagents are Lipofectamine 2000, Lipofectamine 3000, etc. Firstly, A and B solutions are prepared in two centrifuge tubes. A solution is a DNA or RNA solution diluted with serum-free medium, and B solution is a liposome transfection reagent diluted with serum-free medium. Mix solution A and solution B gently and let them stand at room temperature for 15-30 minutes to fully combine and form a complex. The complex is added to the washed cell culture plate, gently shaken, and placed in a culture box for about 6 hours. After 6 hours, the liquid in the culture plate can be aspirated, and complete culture medium can be added to continue cultivation.

Due to the toxic effects of cationic liposomes on cells, in order to prevent the cells from being affected by toxic effects, reasonable experimental conditions should be explored through pre-experiments before conducting formal experiments, including the ratio of liposomes to plasmids, the duration of cell transfection, and the serum content in the culture medium.

Fig. 1 Flow chart of liposome transfection

KMD Bioscience has established a complete set of process technology support for recombinant protein expression and purification, protein fermentation, protein small-scale and pilot scale. The proteins we deliver can be used for drug research, antibody preparation, animal experiments, ELISA, crystal structure studies, in vitro enzyme activity assays, cell experiments, and more. Customers only need to provide us with a protein sequence, CDS or protein name, and we can develop a complete protein expression and purification plan for you in a shorter period of time. In addition, KMD Bioscience has established a comprehensive recombinant protein mammalian expression system, including but not limited to FreeStyle 293-F cell line, Expi 293-F cell line, Expi-CHO-K1, and Expi CHO-S cell lines. Combined with KMD Bioscience's designed mammalian expression vector (containing a full-length CMV promoter and optimized secretion signal peptide sequence), it can provide customers with higher expression levels of recombinant protein mammalian cell expression and mammalian protein customization services.

Reference

[1] Zhu J. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv. 2012;30(5):1158-70.

[2] O'Flaherty R, Bergin A, Flampouri E, et al. Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing. Biotechnol Adv. 2020;43:107552.

[3] Dean DA, Gasiorowski JZ. Liposome-mediated transfection. Cold Spring Harb Protoc. 2011;2011(3):prot5583.