Figure 9.3 Transformation of Eukaryotic Cells
Earlier studies had provided evidence that DNA can transform bacteria. To demonstrate that DNA can also transform eukaryotic cells, researchers isolated mammalian cells that lacked the gene for thymidine kinase. These cells were unable to use thymidine present in growth medium. DNA that contained the gene for thymidine kinase was added to one group of cells, while control DNA that did not contain the thymidine kinase gene was added to another group of cells. Results showed that cells that had taken up the DNA containing the thymidine kinase gene were able to grow in thymidine-containing medium, whereas cells that lacked the thymidine kinase gene were unable to use thymidine present in growth medium and therefore did not grow. These results demonstrated the ability of eukaryotic cells to be transformed by DNA, a process also referred to as transfection. Today, transformation has developed into an essential tool for studying gene function and regulation, and it is also used in medicine for gene therapy, treatment of cancer, and in vaccinations. Further, transformation is key to agricultural biotechnology, allowing insertion of genes for traits such as drought or salt tolerance. Initially, cells were transformed by calcium phosphate co-precipitation; however, efforts to increase transformation efficiency led to the development of other methods that include electroporation, lipid-based reagents, gene gun, and viral vectors. Not every cell type will respond the same to each type of transformation technique. Parameters such as cell density, amount of plasmid DNA, amount of transformation reagent, and culture incubation time must be optimized to maximize transformation efficiency.
Original Paper
Bacchetti, S., and F. L. Graham. 1977. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proceedings of the National Academy of Sciences 74: 1590–1594.
http://www.pnas.org/content/74/4/1590.full.pdf+html
Links
Wikipedia: Transformation
http://en.wikipedia.org/wiki/Transformation_%28genetics%29
Wikipedia: Thymidine kinase
http://en.wikipedia.org/wiki/Thymidine_kinase
Science: Drug Discovery and Biotechnology Trends—Genomics 4: Cellular Invasions
http://www.sciencemag.org/products/ddbt_102204.dtl
University of California, Santa Barbara: New Molecule to Aid Gene Therapy
http://www.ia.ucsb.edu/93106_archived/2006/April03/gene.html
Norrgard, K. 2008 Gene therapy. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Gene-Therapy-760
Chial, H. 2008. Gene-based Therapeutic Approaches. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Gene-Based-Therapeutic-Approaches-3881
Phillips, T. 2008. Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Genetically-Modified-Organisms-GMOs-Transgenic-Crops-and-732
Simmons, D. 2008. Genetic Inequality: Human Genetic Engineering. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Genetic-Inequality-Human-Genetic-Engineering-768