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 a baculovirus to deliver genes to mammalian cells. [1] [2] Baculoviruses are insect viruses that are typically not capable of infecting mammalian cells; however, they can be modified to express proteins in mammalian cells. Unmodified baculoviruses are able to enter mammalian cells; however, their genes are not expressed unless a recognizable mammalian promoter is incorporated upstream of a gene of interest. Both the unmodified baculovirus and its modified counterpart are unable to replicate in humans, making them non-infectious.

Contents

Baculovirus-mediated gene transfer was developed by Dr. Frederick M. Boyce. [3] It has gained widespread use because of certain advantages compared to other transfection methods. [4] [5] [6] BacMam has been found to have some stability and flexibility over other cell line methods, [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 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 insertions of 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, making BacMam 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. Viruses used in the laboratory are unable to replicate in insects, so there is no environmental threat if these particles were to be 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 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.

Uses

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 can be studied using 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 labeling 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 has been performed. [25]

Structural Biology

The BacMam system can be used to produce soluble and membrane-situated glycoproteins for structural studies. [26]

See also

Related Research Articles

<span class="mw-page-title-main">Protein production</span> Biotechnological process

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

<span class="mw-page-title-main">Chinese hamster ovary cell</span> Cell line

Chinese hamster ovary (CHO) cells are a family of immortalized cell lines derived from epithelial cells of the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, and particularly since the 1980s to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

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

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

<span class="mw-page-title-main">Adeno-associated virus</span> Species of virus

Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. They are small replication-defective, nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb).

<i>Baculoviridae</i> Family of viruses

Baculoviridae is a family of viruses. Arthropods, among the most studied being Lepidoptera, Hymenoptera and Diptera, serve as natural hosts. Currently, 85 species are placed in this family, assigned to four genera.

<span class="mw-page-title-main">C3a receptor</span> Protein-coding gene in humans

The C3a receptor also known as complement component 3a receptor 1 (C3AR1) is a G protein-coupled receptor protein involved in the complement system.

Neurturin (NRTN) is a protein that is encoded in humans by the NRTN gene. Neurturin belongs to the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors, which regulate the survival and function of neurons. Neurturin’s role as a growth factor places it in the transforming growth factor beta (TGF-beta) subfamily along with its homologs persephin, artemin, and GDNF. It shares a 42% similarity in amino acid sequence with mature GDNF. It is also considered a trophic factor and critical in the development and growth of neurons in the brain. Neurotrophic factors like neurturin have been tested in several clinical trial settings for the potential treatment of neurodegenerative diseases, specifically Parkinson's disease.

Rhodopsin kinase is a serine/threonine-specific protein kinase involved in phototransduction. This enzyme catalyses the following chemical reaction:

<span class="mw-page-title-main">Baculoviral IAP repeat-containing protein 3</span> Protein-coding gene in the species Homo sapiens

Baculoviral IAP repeat-containing protein3 is a protein that in humans is encoded by the BIRC3 gene.

<span class="mw-page-title-main">OR2K2</span> Protein-coding gene in the species Homo sapiens

Olfactory receptor 2K2 is a protein that in humans is encoded by the OR2K2 gene.

<span class="mw-page-title-main">OR7A5</span> Protein-coding gene in the species Homo sapiens

Olfactory receptor 7A5 is a protein that in humans is encoded by the OR7A5 gene.

<span class="mw-page-title-main">OR51E1</span> Protein-coding gene in the species Homo sapiens

Olfactory receptor 51E1 is a protein that in humans is encoded by the OR51E1 gene.

<span class="mw-page-title-main">GNA11</span> Protein-coding gene in the species Homo sapiens

Guanine nucleotide-binding protein subunit alpha-11 is a protein that in humans is encoded by the GNA11 gene. Together with GNAQ, it functions as a Gq alpha subunit.

A subunit vaccine is a vaccine that contains purified parts of the pathogen that are antigenic, or necessary to elicit a protective immune response. Subunit vaccine can be made from dissembled viral particles in cell culture or recombinant DNA expression, in which case it is a recombinant subunit vaccine.

Heterologous expression refers to the expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment in question. Insertion of the gene in the heterologous host is performed by recombinant DNA technology. The purpose of heterologous expression is often to determine the effects of mutations and differential interactions on protein function. It provides an easy path to efficiently express and experiment with combinations of genes and mutants that do not naturally occur.

The PiggyBac (PB) transposon system employs a genetically engineered transposase enzyme to insert a gene into a cell's genome. It is built upon the natural PiggyBac (PB) transposable element (transposon), enabling the back and forth movement of genes between chromosomes and genetic vectors such as plasmids through a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. The TTAA-specific transposon piggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species, particularly insects. They were discovered in 1989 by Malcolm Fraser at the University of Notre Dame.

<span class="mw-page-title-main">Early 35 kDa protein</span> Anti-apoptotic viral protein

The Early 35 kDa protein, or P35 in short, is a baculoviral protein that inhibits apoptosis in the cells infected by the virus. Although baculoviruses infect only invertebrates in nature, ectopic expression of P35 in vertebrate animals and cells also results in inhibition of apoptosis, thus indicating a universal mechanism. P35 has been shown to be a caspase inhibitor with a very wide spectrum of activity both in regard to inhibited caspase types and to species in which the mechanism is conserved.

<span class="mw-page-title-main">OR1E3</span> Protein-coding gene in the species Homo sapiens

Olfactory receptor family 1 subfamily E member 3 (gene/pseudogene) is a protein that in humans is encoded by the OR1E3 gene.

<span class="mw-page-title-main">Intracellular delivery</span> Scientific research area

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.

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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