Biology:BacMam

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A human mesenchymal stem cell expressing microtubule associated protein fusion to Green fluorescent protein (green) and histone 2b fusion to tagRFP (red) via BacMam gene delivery technology.

Baculovirus gene transfer into Mammalian cells, known from scientific research articles as BacMam, is the use of baculovirus to deliver genes to mammalian cells.[1][2] Baculoviruses are insect viruses that can be modified to express proteins in mammalian cells. The unmodified baculovirus is able to enter those cells; however, its genes are not expressed unless a mammalian recognizable promoter is incorporated upstream of a gene of interest. Both unmodified baculovirus and its modified counterpart are unable to replicate in humans and are thus non-infectious.

Baculovirus-mediated gene transfer was invented by Dr. Frederick M. Boyce.[3] It has gained widespread use because of advantages when compared to other transfection methods.[4][5][6] In addition, BacMam has been found to have inherent flexibility over stable cell lines,[7] 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 the expression of toxic gene products. It has a broad range of transduction including many primary cell types and stem cells.[8] The baculoviral genome has a large capacity for foreign gene insertion with up to 38 kb have been tried successfully.[9] Simultaneous delivery of multiple genes to the same cell is feasible.[10] There are little to no microscopically observable cytopathic effects of BacMam particles on mammalian cells.[11] The level of gene expression can be adjusted by viral dose or chemical additions using histone deacetylase inhibitors.[12] Transduction of cells is performed by liquid-only addition and therefore BacMam is amenable to automated methods. Viruses are stable when stored at 4°C in the dark for long periods of time.[13]

Biosafety considerations

Baculoviruses are Risk Group 1 agents that have been widely used for over 25 years for insect cell protein production applications.[14] 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.[15][16]

Viral entry

Studies on baculovirus entry into human hepatocellular carcinoma cells suggest that BacMam enters mammalian cells via clathrin-mediated endocytosis and possibly via micropinocytosis.[17] Further studies have suggested that caveolae are somehow involved in baculovirus entry in mammalian cells.[18]

Host cell response

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 (HEK293) have revealed that baculovirus transduction is not cytotoxic and does not induce differential transcriptional responses.[19] Similarly, infected Schwann cells retain their characteristic morphological and molecular phenotype and are capable of differentiating in vitro and expressing the P0 myelination marker. Using complementary DNA (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.[20] 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.

  • Bioproduction
BacMam has been used to produce proteins in large quantities using HEK293 cells in a hollow fiber bioreactor system[21]
  • High Throughput Screening
Pharmacology of G protein-coupled receptor is enabled with the use of BacMam technology in drug discovery applications[22]
  • Fluorescence Microscopy
Organelle labeling reagents are commercially available BacMam particles for labeling organelles and other subcellular structures[23]
Single mitochondrion labeled with a mitochondrial targeted green fluorescent protein[24]
  • Receptor Activation/Pathway Analysis
Characterization of serotonin receptor activation via a BacMam delivered GFP fusion to a kinase substrate[25]
  • Structural Biology
BacMam system has been used to produce soluble and membrane glycoproteins for structural studies[26]

See also

  • Baculovirushgv

References

  1. ↑ Hofmann, C; Strauss, M (1998). "Baculovirus-mediated gene transfer in the presence of human serum or blood facilitated by inhibition of the complement system". Gene Therapy 5 (4): 531–536. doi:10.1038/sj.gt.3300607. PMID 9614578. 
  2. ↑ Boyce, FM; Bucher, NL (1996). "Baculovirus-mediated gene transfer into mammalian cells". Proceedings of the National Academy of Sciences of the United States of America 93 (6): 2348–52. doi:10.1073/pnas.93.6.2348. PMID 8637876. Bibcode: 1996PNAS...93.2348B. 
  3. ↑ US patent #5,871,986
  4. ↑ Kost, T; Condreay, JP (2002). "Recombinant baculoviruses as mammalian cell gene-delivery vectors". Trends in Biotechnology 20 (4): 173–180. doi:10.1016/S0167-7799(01)01911-4. PMID 11906750. 
  5. ↑ Kost, Thomas A; Condreay, J Patrick; Jarvis, Donald L (2005). "Baculovirus as versatile vectors for protein expression in insect and mammalian cells". Nature Biotechnology 23 (5): 567–575. doi:10.1038/nbt1095. PMID 15877075. 
  6. ↑ Kost, T; Condreay, J; Ames, R; Rees, S; Romanos, M (2007). "Implementation of BacMam virus gene delivery technology in a drug discovery setting". Drug Discovery Today 12 (9–10): 396–403. doi:10.1016/j.drudis.2007.02.017. PMID 17467576. 
  7. ↑ Davenport, Elizabeth A.; Nuthulaganti, Parvathi; Ames, Robert S. (2009). "BacMam: Versatile Gene Delivery Technology for GPCR Assays". G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology. 552. pp. 199–211. doi:10.1007/978-1-60327-317-6_14. ISBN 978-1-60327-316-9. 
  8. ↑ Zeng, Jieming; Du, Juan; Zhao, Ying; Palanisamy, Nallasivam; Wang, Shu (2007). "Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells". Stem Cells 25 (4): 1055–1061. doi:10.1634/stemcells.2006-0616. PMID 17420229. 
  9. ↑ Cheshenko, N; Krougliak, N; Eisensmith, R C; Krougliak, V A (2001). "A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus". Gene Therapy 8 (11): 846–854. doi:10.1038/sj.gt.3301459. PMID 11423932. 
  10. ↑ Ames, Robert; Nuthulaganti, Parvathi; Fornwald, Jim; Shabon, Usman; Van-Der-Keyl, Harjeet; Elshourbagy, Nabil (2004). "Heterologous Expression of G Protein?Coupled Receptors in U-2 OS Osteosarcoma Cells". Receptors and Channels 10 (3–4): 117–124. doi:10.1080/10606820490515012. PMID 15512846. 
  11. ↑ Cheng, T; Xu, CY; Wang, YB; Chen, M; Wu, T; Zhang, J; Xia, NS (2004). "A rapid and efficient method to express target genes in mammalian cells by baculovirus". World Journal of Gastroenterology 10 (11): 1612–8. doi:10.3748/wjg.v10.i11.1612. PMID 15162535. 
  12. ↑ Pfohl, JL; Worley Jf, 3rd; Condreay, JP; An, G; Apolito, CJ; Kost, TA; Truax, JF (2002). "Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction". Receptors & Channels 8 (2): 99–111. doi:10.1080/10606820212396. PMID 12448791. 
  13. ↑ Jorio, H.; Tran, R.; Kamen, A. (2006). "Stability of Serum-Free and Purified Baculovirus Stocks under Various Storage Conditions". Biotechnology Progress 22 (1): 319–325. doi:10.1021/bp050218v. PMID 16454526. 
  14. ↑ Smith, GE; Summers, MD; Fraser, MJ (1983). "Production of human beta interferon in insect cells infected with a baculovirus expression vector". Molecular and Cellular Biology 3 (12): 2156–65. doi:10.1128/MCB.3.12.2156. PMID 6318086. 
  15. ↑ Kost, T. A., and Condreay, J. P. 2002a. Applied Biosafety 7:167–169.
  16. ↑ 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.
  17. ↑ Matilainen, H.; Rinne, J.; Gilbert, L.; Marjomaki, V.; Reunanen, H.; Oker-Blom, C. (2005). "Baculovirus Entry into Human Hepatoma Cells". Journal of Virology 79 (24): 15452–15459. doi:10.1128/JVI.79.24.15452-15459.2005. PMID 16306616. 
  18. ↑ Long, G.; Pan, X.; Kormelink, R.; Vlak, J. M. (2006). "Functional Entry of Baculovirus into Insect and Mammalian Cells is Dependent on Clathrin-Mediated Endocytosis". Journal of Virology 80 (17): 8830–8833. doi:10.1128/JVI.00880-06. PMID 16912330. 
  19. ↑ Kenoutis, C.; Efrose, R. C.; Swevers, L.; Lavdas, A. A.; Gaitanou, M.; Matsas, R.; Iatrou, K. (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 (8): 4135–4146. doi:10.1128/JVI.80.8.4135-4146.2006. PMID 16571829. 
  20. ↑ Boulaire, Jérôme; Zhao, Ying; Wang, Shu (2009). "Gene expression profiling to define host response to baculoviral transduction in the brain". Journal of Neurochemistry 109 (5): 1203–1214. doi:10.1111/j.1471-4159.2009.06015.x. PMID 19476540. 
  21. ↑ Jardin, B; Zhao, Y; Selvaraj, M; Montes, J; Tran, R; Prakash, S; Elias, C (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 (3): 272–280. doi:10.1016/j.jbiotec.2008.04.006. PMID 18499293. 
  22. ↑ Ames, Robert; Fornwald, James; Nuthulaganti, Parvathi; Trill, John; Foley, James; Buckley, Peter; Kost, Thomas; Wu, Zining et al. (2004). "BacMam Recombinant Baculoviruses in G Protein?Coupled Receptor Drug Discovery". Receptors and Channels 10 (3–4): 99–107. doi:10.1080/10606820490514969. PMID 15512844. 
  23. ↑ Ames, Robert S; Kost, Thomas A; Condreay, J Patrick (2007). "BacMam technology and its application to drug discovery". Expert Opin Drug Discov 2 (12): 1669–1681. doi:10.1517/17460441.2.12.1669. PMID 23488908. 
  24. ↑ Distelmaier, Felix; Koopman, Werner J. H.; Testa, Epifania R.; De Jong, Arjan S.; Swarts, Herman G.; Mayatepek, Ertan; Smeitink, Jan A. M.; Willems, Peter H. G. M. (2008). "Life cell quantification of mitochondrial membrane potential at the single organelle level". Cytometry Part A 73A (2): 129–138. doi:10.1002/cyto.a.20503. PMID 18163486. 
  25. ↑ Huwiler, Kristin G.; MacHleidt, Thomas; Chase, Lucas; Hanson, Bonnie; Robers, Matthew B. (2009). "Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor". Analytical Biochemistry 393 (1): 95–104. doi:10.1016/j.ab.2009.06.018. PMID 19539597. 
  26. ↑ Dukkipati, Abhiram; Park, Hyun Ho; Waghray, Deepak; Fischer, Suzanne; Garcia, Keenan C. (2008). "BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies". Protein Expr Purif 62 (2): 160–170. doi:10.1016/j.pep.2008.08.004. PMID 18782620. 

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