Baculovirus gene transfer into Mammalian cells, known from scientific research articles as BacMam, is the use of baculovirus to deliver genes to mammalian cells. Baculoviruses are insect cell viruses that can be modified to express proteins in mammalian cells. The unmodified baculovirus is able to enter mammalian cells, however its genes are not expressed unless a mammalian recognizable promoter is incorporated upstream of a gene of interest. Both unmodified baculovirus and baculovirus modified with a mammalian promoter are unable to replicate in humans and are thus non infectious. Invented by Dr. Frederick M. Boyce, BacMam is a baculovirus-mediated gene transfer technique that has gained widespread use because of advantages when compared to other transfection methods,. In addition, BacMam has been found to have inherent flexibility over stable cell lines, which has contributed to its adoption as a standard gene transfer technique.
General Properties
The BacMam gene delivery technology is a transient expression system, which facilitates expression of toxic gene products. It has a broad range of transduction including many primary cell types and stem cells. The baculoviral genome has a large capacity for foreign gene insert with up to 38 kb have been tried successfully. Simultaneous delivery of multiple genes to the same cell is feasible. There is little to no microscopically observable cytopathic effects of BacMam particles on mammalian cells. The level of gene expression can be adjusted by viral dose or chemical additions using histone deacetylase inhibitors. Transduction of cells is performed by liquid only addition and therefore BacMam is amenable to automated methods. Finally viruses are stable when stored at 4 °C in the dark for long periods of time.
Biosafety ConsiderationsKost, T. A., and Condreay, J. P. 2002a. Applied Biosafety 7:167–169.Kost, T. A., Condreay, J. P., and Mickelson, C. A. 2000. Bisafety and viral gene transfer vectors. In Biological safety, principles and practices, 3rd ed. D. O. Fleming and D. L. Hunt (eds.), American Society for Microbiology Press, Washington D.C., pp. 579–597.
Baculoviruses are Risk Group 1 agents that have been widely used for over 25 years for insect cell protein production applications. Baculoviruses are produced in insect cells and incapable of replicating in mammalian cells and are not known to cause disease in healthy human adults. Furthermore, BacMam viruses are inactivated by human complement, which reduces risk to researchers. Lastly, viruses used in the laboratory cannot replicate in insects so there is no environmental threat from these particles accidentally being released into the environment.
Viral Entry
Studies on baculovirus entry into human hepatocellular carcinoma cells suggest that BacMam enters mammalian cells via clathrin-mediated endocytosis and possibly via macropinocytosis. Further studies have suggested that caveolae are somehow involved in baculovirus entry in mammalian cells.
To be effective, a gene delivery technology must not interfere with normal cellular function. Cytotoxicity assays and transcriptome analyses on a human HEK cell line have revealed that baculovirus transduction is not cytotoxic and does not induce differential transcriptional responses. Similarly, infected Schwann cells retain their characteristic morphological and molecular phenotype and are capable of differentiating in vitro and express the P0 myelination marker. Using complementary DNAmicroarray technology to examine in vitro and in vivo global cellular gene expression profiles in the rat brain, cultured human astrocytes and human neuronal cells after viral transduction, host antiviral responses were observed. The related genes were mainly those associated with innate immunity, including several of the genes involved in Toll-like receptorsignaling pathway and cytokine-cytokine receptor interaction.
