Generation of Stably Expressing Cell Lines in 28 Days
Generation of Stably Expressing Cell Lines in 28 Days
Altogen Labs provides a stable cell line development service by transforming your cell line to stably express a vector or gene of interest (including RNAi and Tet-inducible cell lines). This is done through the cloning of plasmid DNA that encodes your gene of interest, a correct promoter, and an antibiotic resistance gene. The antibiotic resistance gene is used for the selection of successfully transfected cell lines, as those which have been transfected will exhibit antibiotic resistance and can hence be isolated from the remaining cells.
Stable vs. Transient Transfection
Many research applications may only require simple transient transfections – those that last a day or two – but other long-term studies may need cell lines that have been permanently modified on a genomic level. This is where stable transfections come into play; cells that have been stably transfected will have integrated genetic material in their genome, and will pass it on to future generations. As a result, all future cells will already contain the desired gene, which means that a single flask of stably-transfected cells is enough for an entire research study.
After the cloning of plasmid DNA is complete, transfected genetic material can be expressed in the target cells either transiently or permanently depending on the methods utilized and the experimental questions being investigated. Transient transfections are used most commonly to analyze the short-term impacts of altered gene or protein expression. Plasmid DNA (pDNA) can integrate into host cell line genome and be expressed in a stable manner. Messenger RNA (mRNA) and small RNAs, such as short interfering RNA (siRNA) and microRNA (miRNA), are introduced via the transient transfection process and gene products are expressed in the target cells. However, the nucleic acids do not integrate into the host cell genome. As a consequence, the protein expression is transient and typically results in high expression levels that persist for 24-72 hours when RNA is transfected, or 48-96 hours following transient DNA transfection.
Conversely, in order to analyze the long-term impact of altered gene or protein expression investigators typically utilize stable transfection protocols to develop stable cell lines. Such protocols involve the careful transfection of nucleic acids and additional reagents to ensure nucleic penetration. In order to create stable cell lines, companies take advantage of the small chance that transfected material will naturally be integrated into a cell’s genome. Transfected nucleic acids introduce the gene of interest along with a selectable marker. The growth of transfected cells, in the presence of a selecting agent, will enable the subpopulation of cells where the exogenous genetic material has been incorporated into the genome to persist while the remaining cells undergo selection. Utilizing this method, investigators are able to develop cells that permanently express specific genes through their incorporation in the cellular genome. This method is widely used for protein manufacturing in biotechnology and pharma industry.
Applications and Commercial Details
Many research studies require specially modified cells for drug screening, while industrial applications may need altered protein expression for continued production. Insulin, for example, is manufactured using stably-transfected bacteria that produce insulin at far higher levels than they would naturally. Although stable transfections may not be necessary for all research applications, they are incredibly useful to long-term genetic studies. Researchers can outsource the development of stable cell lines to companies, and receive cells that can then be cultured into a larger population that is used for the study itself. It is important to understand that stable cell lines take some time to generate, as transfection and selection processes require precision and time for completion. Oftentimes, commercial development is far more reliable, as pre-made transfection protocols have already been tested for overall efficiency.
Altogen Labs provides stable cell line development in just 28 days: Receive instant quote for 28-days stable cell line development
Development of Stably Expressing Cell Lines
The development of stably expressing cell lines is a technique used in molecular biology and biotechnology to produce cells that continuously express a specific gene or protein of interest. Stably expressing cell lines are generated by introducing the gene of interest into the cells and selecting for cells that have integrated the gene into their genome.
The development of stably expressing cell lines typically involves the following steps:
- Selection of cell line: A suitable cell line is selected based on its growth characteristics and suitability for the intended application.
- Design of expression vector: An expression vector is designed that contains the gene of interest under the control of a suitable promoter and selection marker.
- Transfection of cells: The expression vector is introduced into the cells using a transfection method, such as electroporation or chemical transfection.
- Selection of stably expressing cells: Cells that have integrated the expression vector into their genome are selected by applying selection pressure, such as treatment with a drug that is only tolerated by cells that express the selection marker.
- Clonal expansion: Individual cells are isolated and expanded to form clonal cell lines that stably express the gene of interest.
- Characterization of stably expressing cells: The stably expressing cells are characterized by measuring the level and duration of gene expression, and by confirming that the gene of interest is expressed in a functional form.
Stably expressing cell lines are important tools for studying gene function, screening for drug candidates, and producing recombinant proteins or antibodies for therapeutic or diagnostic purposes. The development of stably expressing cell lines can be a time-consuming process that requires careful optimization of transfection and selection conditions, as well as appropriate controls to ensure the reliability and reproducibility of the results. Altogen Labs provides stable cell line development in just 28 days: Receive instant quote for 28-days stable cell line development
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