High Five (BTI-Tn-5B1-4) is an insect cell line that originated from the eggs of the cabbage looper, Trichoplusia ni. [1] It was developed by the Boyce Thompson Institute for Plant Research.
High Five cells have become one of the most commonly used cell lines for recombinant protein expression using baculovirus or transfection, and have been demonstrated to express more recombinant protein than other lepidopteran cell lines, such as Sf9 cells. [2] [3] [4] [5] [6] [7] [8] The High Five cells have been used to produce the VLP-based HPV vaccine Cervarix. [9]
They can be grown in the absence of serum, and can be cultured in a loose attached state or in suspension [10] High Five cells produce abundant microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs), making it suitable to study all three types of small silencing RNAs. [11]
The High Five cell line, like other cell lines, [12] has been found to host a non-pathogenic adventitious alphanodavirus. Researchers at the Boyce Thompson Institute were able to isolate virus-free sub-clones, named Tnao38 [13] [14] and Tnms42. [15]
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.
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.
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.
Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, are an immortalised cell line derived from HEK cells isolated from a female fetus in the 1970s.
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
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.
Pharming, a portmanteau of farming and pharmaceutical, refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO). Pharming is also known as molecular farming, molecular pharming, or biopharming.
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.
Vero cells are a lineage of cells used in cell cultures. The 'Vero' lineage was isolated from kidney epithelial cells extracted from an African green monkey. The lineage was developed on 27 March 1962 by Yasumura and Kawakita at the Chiba University in Chiba, Japan. The original cell line was named Vero after an abbreviation of verdareno, which means 'green kidney' in Esperanto, while vero itself means 'truth' in Esperanto.
Sf21 is a continuous cell line developed from ovaries of the Fall Army worm, Spodoptera frugiperda, a moth species that is an agricultural pest on corn and other grass species. It was originally developed in the United States at the Henry A. Wallace Beltsville Agricultural Research Center. Sf9 is a substrain (clone) of these cells that was isolated from Sf21 by researchers at Texas A&M University.
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.
Agroinfiltration is a method used in plant biology and especially lately in plant biotechnology to induce transient expression of genes in a plant, or isolated leaves from a plant, or even in cultures of plant cells, in order to produce a desired protein. In the method, a suspension of Agrobacterium tumefaciens is introduced into a plant leaf by direct injection or by vacuum infiltration, or brought into association with plant cells immobilised on a porous support, whereafter the bacteria transfer the desired gene into the plant cells via transfer of T-DNA. The main benefit of agroinfiltration when compared to the more traditional plant transformation is speed and convenience, although yields of the recombinant protein are generally also higher and more consistent.
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.
Baculovirus gene transfer into Mammalian cells (BacMam) is the use of a baculovirus to deliver genes to mammalian cells. 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.
A genetically modified virus is a virus that has been altered or generated using biotechnology methods, and remains capable of infection. Genetic modification involves the directed insertion, deletion, artificial synthesis or change of nucleotide bases in viral genomes. Genetically modified viruses are mostly generated by the insertion of foreign genes intro viral genomes for the purposes of biomedical, agricultural, bio-control, or technological objectives. The terms genetically modified virus and genetically engineered virus are used synonymously.
Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), are commonly used in insect cell culture for recombinant protein production using baculovirus. They were originally established from ovarian tissue. They can be grown in the absence of serum, and can be cultured attached or in suspension.
The use of insect cell lines as production hosts is an emerging technology for the production of bio pharmaceuticals. There are currently more than 100 insect cell lines available for recombinant protein production with lines derived from Bombyx mori, Mamestra brassicae, Spodoptera frugiperda, Trichoplusia ni, and Drosophila melanogaster being of particular interest. Insects cell lines are commonly used in place of prokaryotic ones because post-translational modifications of proteins are possible in insect cells whereas this mechanism is not present in prokaryotic systems. The Sf9 cell line is one of the most commonly used lines in insect cell culture.
Flock House virus (FHV) is in the Alphanodavirus genus of the Nodaviridae family of viruses. Flock House virus was isolated from a grass grub at the Flock House research station in Bulls, New Zealand. FHV is an extensively studied virus and is considered a model system for the study of other non-enveloped RNA viruses owing to its small size and genetic tractability, particularly to study the role of the transiently exposed hydrophobic gamma peptide and the metastability of the viral capsid. FHV can be engineered in insect cell culture allowing for the tailored production of native or mutant authentic virions or virus-like-particles. FHV is a platform for nanotechnology and nanomedicine, for example, for epitope display and vaccine development. Viral entry into host cells occurs via receptor-mediated endocytosis. Receptor binding initiates a sequence of events during which the virus exploits the host environment in order to deliver the viral cargo in to the host cytosol. Receptor binding prompts the meta-stability of the capsid–proteins, the coordinated rearrangements of which are crucial for subsequent steps in the infection pathway. In addition, the transient exposure of a covalently-independent hydrophobic γ-peptide is responsible for breaching cellular membranes and is thus essential for the viral entry of FHV into host cells.
Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells with a chemical delivery agent like calcium phosphate (CaPi) or polyethyleneimine (PEI). However, unlike "stable expression," the foreign DNA does not fuse with the host cell DNA, resulting in the inevitable loss of the vector after several cell replication cycles. The majority of transient gene expressions are done with cultivated animal cells. The technique is also used in plant cells; however, the transfer of nucleic acids into these cells requires different methods than those with animal cells. In both plants and animals, transient expression should result in a time-limited use of transferred nucleic acids, since any long-term expression would be called "stable expression."
Max Duane Summers is an American molecular biologist and inventor, known for his work on the Baculovirus Expression Vector System (BEVS).