TRANSFECTION: NON-VIRAL VS VIRAL
With in vivo transfection technology, scientists are able to insert exogenous DNA into a cell that alters or changes coding DNA, non-coding DNA or cellular function. The end result enables biomedical scientists and researchers to study complex cellular processes and diseases. In vivo delivery reagents and kits are manufactured by Altogen Biosystems.
Existing in a scientists toolbox are both non-viral (lipid-, polymer-, nanoparticle-based, etc) and viral transfection methods. Viral vectors are usually chosen for high delivery efficiency, however, concerning factors include their safety in patients, toxicity (i.e. minimizing the amount of changes it makes to the cell), stability (maintaining a constant genome in the virus), specificity (i.e. ensure that the transfection only occurs in the desired cells) and identification (i.e. vectors must incorporate marker genes for identification). Types of viral vectors used include: retroviruses, lentiviruses and adenoviruses.
For transfection of suspension cell lines, as well as primary cells, an electroporation technique is used as effective means to enable intracellular delivery. This method utilizes an electrical field to open pores in the membrane of cells; thus, providing transport of charged molecules to the cellular cytoplasm and nuclei.
Also, hybrid transfection methods were developed including virosomes and other mixtures of virus vectors with particles/non-DNA molecules.
APPLICATIONS OF DNA TRANSFECTION
Transfection in an important technique that influenced many disease treatments, including cancer and diabetes. Insulin producing genes have allowed scientists to create cell lines that produce the protein coding for human insulin, thus, providing a source for pure medical grade insulin. This is just one of many examples of medical treatments that have been created using this technology.
Stable transfection, often called permanent transfection, is insertion of plasmid DNA into the genome of cancer cell DNA. Creating a stable cell line allows a researcher to quantitate long term effects of the introduced gene. Downstream studies utilizing the stable cell line includes overexpressing the gene insert for protein production, observe results of cell based assays to determine effects on apoptosis or incorporation of a fluorescent marker to track tumor growth when implanted in an immunodeficient mouse xenograft model (i.e. CDX mouse model).
An important aspect of creating a stable cell line is to make sure the plasmid backbone contains an antibiotic resistance gene so that a positively transfected cell can be selected from the population of treated cells. The transfected cells are maintained under constant antibiotic selection so that the cell population is forced into a majority of positively expressing cells.
The length of efficacy from transfecting DNA is the benefit of performing a stable transfection versus a transient transfection. Small interfering RNA (i.e. siRNA, miRNA) typically exhibit 48 hour maximal target effect due to the transient transfection. However, the transfection of plasmid DNA into a cell, followed by integration of the DNA into the cellular genome, results in expression of the target gene indefinitely.