Cell lines are populations of cells grown and maintained in a laboratory, often indefinitely, under specific conditions. Transfection is a technique that introduces foreign genetic material, such as DNA or RNA, into these cells. This process allows scientists to study gene function and protein expression. Following transfection, screening identifies and isolates cells that have successfully incorporated and are expressing the new genetic material. This combination of transfection and screening is important in biotechnology and research.
Understanding Transfection and Its Purpose
Transfection introduces nucleic acids, like DNA or RNA, into eukaryotic cells using non-viral methods. This gene transfer technology enables the study of gene function and protein expression. Scientists perform transfection to produce specific proteins, study gene function, or develop new therapeutic strategies.
The introduced genetic material may remain in the cell temporarily, a process called transient transfection, where it is expressed for a limited time and does not replicate. Alternatively, the genetic material can stably integrate into the recipient cell’s genome, replicating alongside the host genome, which is known as stable transfection. Stable transfection allows for long-term gene expression studies or the creation of cell lines with altered characteristics for future experiments.
Why Screening is Essential
Transfection is not always efficient, as only a fraction of cells may successfully take up the foreign genetic material. The process can also be imprecise, with the genetic material integrating randomly into the host genome in stable transfection, potentially affecting its expression or the cell’s normal functions. For example, stable integration of a plasmid into the genome is a rare event, occurring in approximately 1 in 10,000 to 1 in 10 million cells, depending on the cell line.
Cells might not express the newly introduced genetic material effectively, even if integrated. This variability necessitates a subsequent screening step to identify and isolate the desired cells from the larger population of untransfected or unsuccessfully transfected cells. Without screening, it would be difficult to obtain reliable experimental results with a pure population of cells exhibiting the desired genetic modification.
Key Screening Methods
Various methods are employed to identify cells that have successfully taken up and are expressing new genes.
Selection Markers
One common approach uses genes that provide a selective advantage to the transfected cells, often by conferring resistance to certain drugs. These “selection markers” are typically introduced simultaneously with the gene of interest. For instance, an antibiotic resistance gene can be included in the genetic material being introduced.
When the cells are grown in a medium containing that specific antibiotic, only the cells that have successfully integrated and expressed the resistance gene, along with the gene of interest, will survive and proliferate. This method eliminates untransfected cells, allowing for the isolation of a population enriched for the desired genetic modification. Examples of such markers include genes conferring resistance to antibiotics like G-418 or blasticidin.
Reporter Genes
Reporter genes produce a detectable signal. These genes are linked to the gene of interest or placed under the control of a specific promoter. When the gene of interest is expressed, the reporter gene is also expressed, producing a visible signal like fluorescence or a color change. Commonly used reporter genes include green fluorescent protein (GFP), which makes cells glow green under specific light, or luciferase, an enzyme that produces light when a substrate is added. The signal from the reporter gene allows researchers to identify and quantify successfully transfected cells, or assess the strength of gene expression.
Functional Assays
Functional assays directly measure the desired biological activity or protein production resulting from transfection. These assays confirm the introduced gene is not only present but also actively functioning as intended. For example, if a gene for a specific enzyme was introduced, a functional assay would measure the enzyme’s activity in the cell extract.
Other functional assays might assess changes in cell growth, signaling pathways, or the production of a therapeutic protein. Techniques like quantitative PCR (qPCR) can measure new messenger RNA expression levels, while Western blotting or ELISA can detect and quantify the protein product. These assays provide direct evidence of successful gene expression and its downstream effects.
Applications of Screened Cell Lines
Successfully screened cell lines are valuable tools across various scientific and medical fields. These specialized cell lines, with their genetic modifications, advance research and development.
In drug discovery and development, screened cell lines test new drug candidates. They can model specific diseases or biological pathways, allowing researchers to evaluate how compounds interact with target proteins or cellular processes, speeding up the identification of potential therapeutics. For example, the NCI-60 project screens 60 human tumor cell lines to identify compounds with anticancer activity.
Screened cell lines are also employed in biopharmaceutical production. These engineered cells can be grown in large quantities to manufacture therapeutic proteins, antibodies, or vaccines. Chinese Hamster Ovary (CHO) cells, for instance, are frequently transfected with genes encoding therapeutic proteins, which are then harvested and purified for medical use.
Modified cell lines are also used for disease modeling, allowing scientists to create cellular models that mimic human diseases. By introducing or altering specific genes, researchers can study disease mechanisms, identify new drug targets, and test potential treatments in a controlled environment. This includes gene therapy research, where screened cell lines develop and test strategies for correcting genetic defects at a cellular level.