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BacMam is the use of baculovirus to deliver genes to mammalian cells.[1][2] 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 (BacMam) are unable to replicate in humans and are thus non infectious.
BacMam is a gene delivery technology that has been shown to have advantages when compared to other transfection methods.[3] Existing transfection methods suffer from cytotoxic effects, biosafety concerns and reagent expenditures. While BacMam has its limitations it does overcome many of the issues of other gene delivery technologies.[4]
General Properties
- Transient expression, facilitates expression of toxic gene products
- Broad host cell range with transduction of many primary cell types and stem cells[5]
- Large insert capacity, 38 kb[6]
- Simultaneous delivery of multiple genes[7]
- Little to no microscopically observable cytopathic effect
- Expression levels are can be adjusted by virus dose or chemical addition[8]
- Cell transduction by liquid addition only and therefore amenable to automated methods
- Viruses stable when stored at 4ºC in the dark
Biosaftey Considerations
- Baculoviruses are Risk Group 1 agents
- Used widely for over 25 years for insect cell protein expression[9]
- Produced in insect cells and incapable of replicating in mammalian cells
- Not known to cause disease in healthy human adults
- BacMam viruses are inactivated by human complement
- Viruses used in the laboratory cannot replicate in insects so there is no environmental threat
Viral Entry
Studies on baculovirus entry into human hepatocellular carcinoma cells suggest that BacMam enters mammalian cells via clathrinmediated endocytosis and possibly via macropinocytosis.[10] Further studies have suggested that caveolae are somehow involved in baculovirus entry in mammalian cells.[11]
Host Cell Response
To be effective, a gene delivery technology must not interfere with normal cellular function. Toxicity assays and transcriptome analyses on a human cell line (HEK293) have revealed that baculovirus transduction is not cytotoxic and does not induce differential transcriptional responses.[12] 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 cDNA microarray 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.[13] The related genes were mainly those associated with innate immunity, including several of the genes involved in Toll-like receptor signaling pathway and cytokine-cytokine receptor interaction.
Applications
- Bioproduction
- BacMam has been used to produce proteins in large quantities using HEK293 cells in a hollow fiber bioreactor system[14]
- High Throughput Screening
- Pharmacology of G protein-coupled receptor is enabled with the use of BacMam technology in drug discovery applications[15]
- Fluorsecence Microscopy
- Organelle Lights™ reagents are commercially available BacMam particles for labeling organelles and other subcellular structures[16][17]
- Receptor Activation/Pathway Analysis
- Characterization of serotonin receptor activation via a BacMam delivered GFP fusion to a kinase substrate[18]
See also
References
- ^ Hofmann, Christian et al(1995) Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc. Nat. Acad. Sci. USA 92:10099-10103.
- ^ Boyce, Frederick M. & Bucher, Nancy L. R. (1996) Baculovirus-mediated gene transfer into mammalian cells. Proc. Nat. Acad. Sci. USA 93:2348-2352.
- ^ Kost, Thomas A. et al (2007) Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discovery Today Volume 12, Numbers 9/10.
- ^ Kost, Thomas A. et al (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology 23:567-575.
- ^ Zeng, J. et al (2007) Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells. Stem Cells 25:1055–1061.
- ^ Cheshenko, C. et al (2001) A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Therapy 8:846–854.
- ^ Ames, Robert A. (2004) Heterologous Expression of G Protein–Coupled Receptors in U-2 OS Osteosarcoma Cells. Receptors and Channels 10: 117–124.
- ^ Pfohl, Jeffery L. et al (2002) Titration of KATP Channel Expression in Mammalian Cells Utilizing Recombinant Baculovirus Transduction. Receptors and Channels 8:99–111.
- ^ Smith, Gale E. et al(1983) Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector. Molecular and Cellular Biology 3:2156-2165.
- ^ Matilainen, Heli et al (2005) Baculovirus Entry into Human Hepatoma Cells. Journal of Virology 79:15452–15459.
- ^ Long, Gang et al (2006) Functional Entry of Baculovirus into Insect and Mammalian Cells Is Dependent on Clathrin-Mediated Endocytosis. Journal of Virology 80:8830–8833.
- ^ Kenoutis, Christos et al (2006) Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression. Journal of Virology 80:4135–4146.
- ^ Boulaire, Jerome (2009) Gene expression profiling to define host response to baculoviral transduction in the brain. Journal of Neurochemistry 109:1203-1214.
- ^ Jardin, B. A. et al (2008) Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system. Journal of Biotechnology 135:272-280.
- ^ Ames, Robert S. et al (2004) BacMam Recombinant Baculoviruses in G Protein–Coupled Receptor Drug Discovery. Receptors and Channels 10:99–107.
- ^ Ames, Robert S. et al (2007) BacMam technology and its application to drug discovery. Expert Opin. Drug Discov. 2:1669-1681.
- ^ Organelle Lights™
- ^ Huwiler, K. G. et al (2009) Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor. Analytical Biochemistry 393:95-104.