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. HMSC MAP4GFP H2BRFP.jpg
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 (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.

Contents

Baculovirus-mediated gene transfer was developed 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 the insertion of foreign genes, with up to 38 kb having been successfully tested. [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.

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

See also

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<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

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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. Bibcode:1996PNAS...93.2348B. doi: 10.1073/pnas.93.6.2348 . PMC   39799 . PMID   8637876.
  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. PMC   3610534 . 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. Vol. 552. pp. 199–211. doi:10.1007/978-1-60327-317-6_14. ISBN   978-1-60327-316-9. PMID   19513651. S2CID   21324231.
  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 . PMC   4572764 . 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.{{cite journal}}: CS1 maint: numeric names: authors list (link)
  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. S2CID   41069508.
  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. PMC   370086 . 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. PMC   1316037 . 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. PMC   1563848 . 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. PMC   1440473 . 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; Romanos, Michael (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. S2CID   45039136.
  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. S2CID   5147755.
  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. PMC   2637115 . PMID   18782620.
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