The 293T cell line is a commonly used cell line in molecular biology research, derived from human embryonic kidney cells. The "T" in 293T denotes that these cells have been transformed with the SV40 large T antigen, which allows them to grow indefinitely in culture. This cell line is particularly useful for expressing high levels of recombinant proteins due to its high transfection efficiency and ability to support the replication of a wide range of vectors, including those used in transient and stable expression systems.
Characteristics of 293T Cells
293T cells are known for their robust growth characteristics, making them an ideal choice for large-scale protein production. They can be cultured in a variety of media and are often used in bioreactors for the production of therapeutic proteins and vaccines. The SV40 large T antigen expressed in these cells enables them to bypass normal cell cycle checkpoints, contributing to their immortalization and high proliferation rate. This characteristic, however, also means that 293T cells must be handled with caution, as they can potentially form tumors if injected into animals.
Applications of 293T Cells in Research
Due to their versatility and high transfectability, 293T cells are widely used in various research applications, including the production of recombinant proteins for structural studies, the development of gene therapy vectors, and the study of viral replication and pathogenesis. They are also used in the production of adenoviral vectors for gene therapy and vaccine development. The ability to easily manipulate these cells genetically makes them a valuable tool for studying the function of specific genes and their products in a controlled cellular environment.
| Characteristic | Description |
|---|---|
| Growth Medium | Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics |
| Transfection Efficiency | High, suitable for transient and stable expression of recombinant proteins |
| Growth Rate | Rapid, with a doubling time of approximately 24 hours |
| Cell Morphology | Epithelial, adherent cells |
Stable Expression in 293T Cells
Stable expression in 293T cells involves the integration of the gene of interest into the host cell genome, allowing for long-term production of the recombinant protein. This is achieved through the use of vectors that contain selectable markers, such as antibiotic resistance genes, which enable the selection of cells that have successfully integrated the vector. The process of establishing a stable cell line involves transfecting the cells with the expression vector, selecting the transfected cells using antibiotics, and then cloning and characterizing individual cell clones to identify those with the highest expression levels of the desired protein.
Considerations for Stable Expression
Several factors must be considered when establishing a stable expression cell line in 293T cells, including the choice of vector, the promoter used to drive gene expression, and the conditions used for cell culture and selection. The use of strong promoters, such as the CMV promoter, can drive high levels of gene expression, but may also lead to promoter silencing over time. Therefore, careful optimization of the expression system is necessary to achieve stable, long-term production of the recombinant protein.
The stability of expression over time is also influenced by the site of vector integration into the host genome. Some genomic locations may be more prone to gene silencing due to epigenetic modifications, while others may support long-term, high-level expression. Techniques such as fluorescence-activated cell sorting (FACS) can be used to enrich for cell populations with high expression levels, further stabilizing protein production.
What are the advantages of using 293T cells for recombinant protein production?
+293T cells offer several advantages for recombinant protein production, including high transfection efficiency, rapid growth rate, and the ability to perform complex post-translational modifications. These characteristics make them an ideal choice for producing large quantities of therapeutic proteins and vaccines.
How are stable expression cell lines established in 293T cells?
+Stable expression cell lines are established through the transfection of 293T cells with an expression vector containing the gene of interest and a selectable marker. Transfected cells are then selected using antibiotics, and individual cell clones are characterized to identify those with the highest expression levels of the desired protein.
In summary, 293T cells are a powerful tool for recombinant protein production and research, offering high transfection efficiency, rapid growth, and the ability to perform complex post-translational modifications. Their use in stable expression systems allows for the long-term production of therapeutic proteins and vaccines, making them a crucial component in the development of new treatments for a variety of diseases.
