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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.
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.
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]
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]
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]
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 can be studied using 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 has been performed. [25]
The BacMam system can be used to produce soluble and membrane-situated glycoproteins for structural studies. [26]
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.
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.
Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. Both in-vivo assembly and in-vitro assembly have been successfully shown to form virus-like particles. VLPs derived from the Hepatitis B virus (HBV) and composed of the small HBV derived surface antigen (HBsAg) were described in 1968 from patient sera. VLPs have been produced from components of a wide variety of virus families including Parvoviridae, Retroviridae, Flaviviridae, Paramyxoviridae and bacteriophages. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells.
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).
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.
Viral vectors are modified viruses designed to deliver genetic material into cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, agriculture, and medicine.
Rhodopsin kinase is a serine/threonine-specific protein kinase involved in phototransduction. This enzyme catalyses the following chemical reaction:
Baculoviral IAP repeat-containing protein3 is a protein that in humans is encoded by the BIRC3 gene.
Baculoviral IAP repeat-containing protein 2 is a protein that in humans is encoded by the BIRC2 gene.
Receptor-type tyrosine-protein phosphatase PCP-2, is an enzyme that in humans is encoded by the PTPRU gene.
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.
High Five (BTI-Tn-5B1-4) is an insect cell line that originated from the ovarian cells of the cabbage looper, Trichoplusia ni. It was developed by the Boyce Thompson Institute for Plant Research.
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.
Olfactory receptor family 1 subfamily E member 3 (gene/pseudogene) is a protein that in humans is encoded by the OR1E3 gene.
Helicoverpa zea nudivirus 2 is an enveloped, rod-shaped, nonoccluded, double stranded DNA (dsDNA) sexually transmitted virus whose natural host is the corn earworm moth. At about 440 by 90 nm, it is the causative agent of the only sexually transmitted viral disease of any insect. It was originally identified in a colony of corn earworm moths established and maintained in Stoneville, Mississippi, U.S. and was found to be responsible for the sterility of those infected.
Max Duane Summers is an American molecular biologist and inventor, known for his work on the Baculovirus Expression Vector System (BEVS).
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.
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