A plasmid is a small, circular piece of DNA that exists separately from the main chromosomal DNA in bacteria and some other microscopic organisms. These molecules can replicate independently within a host cell. Transfection is the process of introducing foreign DNA, such as a plasmid, into eukaryotic cells (cells with a nucleus). This allows scientists to modify cell genetic information to study gene function or produce specific proteins.
The Plasmid’s Blueprint
Plasmids are engineered with specific components. The origin of replication (ori) is a DNA sequence that enables the plasmid to be copied within the host cell, ensuring its propagation.
Plasmids also include a selectable marker, typically a gene providing antibiotic resistance. This marker allows scientists to identify cells that have successfully taken up the plasmid, as only these cells will survive in the presence of the corresponding antibiotic. A multiple cloning site (MCS) is another important feature, containing several unique recognition sites for restriction enzymes. These sites allow scientists to precisely insert a gene of interest into the plasmid.
Introducing Plasmids into Cells
Transfection is the process of introducing plasmids into cells, particularly eukaryotic cells. It involves overcoming the cell membrane to allow foreign DNA to enter. Various methods achieve this temporary disruption and DNA entry, broadly categorized as chemical or physical techniques.
Chemical methods often use reagents like liposomes, tiny vesicles similar to cell membranes. In lipofection, cationic (positively charged) lipids mix with negatively charged DNA to form complexes called lipoplexes. These lipoplexes bind to the cell membrane and enter the cell, often via endocytosis.
Physical methods directly create temporary openings in the cell membrane. Electroporation applies an electrical pulse to cells and plasmids, temporarily destabilizing the cell membrane and creating pores for DNA entry. These pores reseal once the pulse is removed, allowing the cell to resume normal functions.
Why and How Transfected Plasmids Are Used
Transfected plasmids are widely used in scientific and medical fields to introduce new genetic instructions into cells. A primary application is in gene expression studies, where researchers use plasmids to produce specific proteins in cultured cells. This allows for studying protein function, structure, and interactions, and generating desired proteins for research or therapeutic purposes, such as human insulin.
In gene therapy research, transfected plasmids deliver therapeutic genes into cells to treat or prevent diseases. This approach aims to correct genetic defects or introduce new functions to combat conditions like cancer or inherited disorders. For instance, plasmids can overexpress genes that produce anti-tumor agents or immune-stimulating factors.
Plasmids also play a role in vaccine development, particularly DNA vaccines. These vaccines use plasmids carrying genes that encode viral antigens, prompting transfected cells to produce antigens and trigger an immune response. This method has shown promise in preclinical models for infectious diseases, including COVID-19. Furthermore, transfected plasmids are instrumental in creating genetically modified organisms by introducing new traits or altering existing ones, contributing to advancements in agriculture and biotechnology.
What Happens After Transfection
Once a plasmid enters a cell through transfection, its fate can vary, leading to either transient or stable expression. In transient transfection, the plasmid remains separate from the host cell’s chromosomes. The genes on the plasmid are expressed for a limited time, typically a few days (24-96 hours), because the plasmid does not replicate with the host cell’s DNA and is gradually lost as the cells divide. This method is often chosen for short-term experiments or rapid protein production.
Conversely, stable transfection involves the integration of the plasmid’s DNA into the host cell’s genome. This integration means that the foreign gene becomes a permanent part of the cell’s genetic material and is replicated along with the cell’s own chromosomes during cell division. As a result, the introduced gene is consistently expressed across many cell generations, leading to a stably transfected cell line. After successful entry and, in some cases, integration, the cell’s machinery reads the instructions on the plasmid. This involves transcription, where the DNA is converted into messenger RNA (mRNA), followed by translation, where the mRNA sequence is used to assemble the desired protein.