Transient Transfection Services

Depending on the temporal and spatial differences in protein expression, the transfection can be categorized into stable and transient transfection. In transient transfection, the DNA plasmid containing the foreign genes enter the cells but do not integrate it into their genome. Compared with stable transfection, which is more suitable for long-term protein production, transient transfection is preferred option for small scale protein production as it allows for rapid protein expression.

Biologics International Corp. (BIC) offers transient transfection services, including short-term transient expression of genes and small-scale protein production for research and development purpose. Using our developed and optimized in-house transfection reagents, we have ability to produce large quantities of proteins/antibodies efficiently. For further information please contact us, we will be happy to assist you.

Transient Transfection Protocol

Codon Optimization & Gene Synthesis

The comprehensive transient transfection service of BIC can be initiated from gene synthesis. Our self-designed MaxCodonTM software can optimize secondary structure of codon mRNA and ribosome binding sites to improve the efficiency of protein expression. Then, the plasmid DNAs encoding the protein of interest and antibiotic-resistant gene can be constructed.

Cell Recovery

Before transfection, cell recovery is generally the first step, which is the process of thawing and re-culture of stored cells. When frozen tubes are taken out from the liquid nitrogen or refrigerator, they must be quickly placed in a preheated water bath (37 ℃-40 ℃). After the liquid is completely melted in the frozen tubes, the cells are transferred to the centrifuge tube with culture medium. Then, the cells are replated in a culturing bottle and cultivated using the conventional method.

Transfection

The cultured cells are inoculatde into 6-wells plates and left to stand so that 80% of the cells form a monolayer at the bottom of the plate. Then, the cells are washed with serum-free medium, and a mixed solution containing the plasmid and transfection reagent is added. After adding culture medium, the culturing of cells is continued. Furthermore, we have several other transfection methods such as electroporation.

Expression & Quality Control

Depending on the construct used, the transient transfected cells are harvested after 24-96 hours of transfection. Using our optimized transfection method, we can effectively prolong the life cycle of host cells and increase the accumulation time of target proteins. These cells can express the target proteins for 5-7 days, followed by protein purification.

Purity assay of protein/antibody can be performed using SDS-PAGE or HPLC (optional). Concentration assay of protein and antibody is performed using BCA/Bradford and A280 methods, respectively. LC-MS/MS peptide mapping is used to determine protein identity if necessary.

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Transient Transfection of Cells

Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells 293 are the generally used mammalian cells in protein production. The proteins that are expressed in two common cells exhibit extremely significant differences in their glycosylation pattern. The glycoprotein expressed by HEK cells is theoretically closer to the structure of human proteins [1]. Several human proteins (such as coagulation factor and receptor proteins) are primarily expressed by HEK cells [2]. However, CHO cells are the most commonly used mammalian cell line for the mass production of therapeutic proteins as they can produce recombinant protein on the scale of 3-10 g/L of culture [3].

Transient Transfection Methods

Transfection methods include chemical and physical methods, such as calcium phosphate exposure, lipofection, electroporation, microinjection, and gene gun. All the methods must allow delivering DNA molecules through the cellular membrane without any permanent damage to the cells.

Chemical-based transfection methods are the most commonly used due to the ease and cost of transfection reagents. Lipofection is a transfection method using liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer, and are able to release their contents.

Physical-based methods such as electroporation are procedures using high-voltage electric shocks to introduce DNA into cells. Due to disruption of the cellular membrane, this method is an appropriate option for cells that have been traditionally difficult to transfect. In addition, the supercoiled plasmid DNA appears more efficient in transfection.

Transfection Methods	Advantages	Disadvantages
Calcium phosphate exposure	Easy for operation	High requirement for DNA concentration Not suitable for primary cells
Cationic lipofection	Easy for operation Good method for the majority of cells	Relatively high requirement for DNA concentration Toxicity to cells
Electroporation	Suitable for different types of DNA fragments	Optimized for different cells High mortality rate
Microinjection	Good for engineering modification & establishment of transgenic animals	Complex for operation Exogenous gene integration sites and copy number cannot be controlled

The Difference between Transient and Stable Transfection

Mammalian cells can express correctly folded and post-translational modified proteins. There are two types of transfection of mammalian cells, known as transient transfection and stable transfection. Stable transfection can be used to produce large amounts of protein in the long-term by introducing the DNA into the chromosome of cells, while transient transfection can be used to express the gene of interest rapidly and efficiently. Although this mechanism of protein production is short lived, it has the benefite of advancing our understanding of basic biology, such as the functional significance of genes.

Cell Transfection	Transient Transfection	Stable Transfection
Timeline of expression	Rapid (3-4 weeks)	Lengthy (12-18 weeks)
Cost	Relatively inexpensive	Expensive
Expression level	Relatively low	High and stable
Foreign gene	Lost in few days	Exists over a long period of time
Secreted or membrane proteins	Good to yield	Good to yield
Intracellular proteins	Hard to yield	Hard to yield
Folding and post-translational modifications	Yes	Yes

References

1.Amelie Croset, Laurence Delafosse, et al. Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells. Journal of Biotechnology, vol.161, 2012, pp.336-348.

2.Jennifer Dumont, Don Euwart, et al. Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, vol.36, 2016, pp.1110-1122.

3.Florian M wurm, David Hacker. First CHO genome. Nature Biotechnology, vol.29, 2011, pp.718-720.

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