Transient expression

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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). [1] 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. [2] 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."

Contents

Methodology varies depending on the organism to transform. While plants can be transformed with a construct introduced into Agrobacterium tumefaciens via agroinfiltration or floral dip, most animal cells would require a viral vector. In humans, the field of transient transformation advanced rapidly during the 2020–2021 COVID-19 pandemic with major COVID-19 vaccines using either direct mRNA transfer into human or adenovirus vectors, with the RNA being expressed in the host human to produce spike proteins that induce an immune response.

Advantages

When choosing between inducing transient or stable expression in cells, time frame and experimental goal must be taken into consideration. Transiently transfected cells are often used to study the effects of short-term gene expression, perform RNA interference (RNAi)‑mediated gene silencing, or quickly generate small-scale recombinant proteins. [3] This rapid generation small quantities of recombinant proteins can be applied towards evaluating their potential as drug candidates or examining their integrity of constructs during stages of vector development. Additionally, transient expression can be a useful tool when aiming to optimize selected parameters before undergoing the time-consuming process of scale-up in stably transfected cells. [4] Typically, the cells are harvested within 1-4 days after successful transfection. For even quicker results, replacing DNA with mRNA can result in transient expression within minutes after successful transfection in some systems; this process bypasses translocation to the nucleus and transcription. [5]

If stable, long-term gene expression is desired, stable transfection of cells is more useful. However, since successful integration of a DNA vector into the chromosome is a rare occurrence, this process is more difficult and time-consuming, and is reserved for large-scale protein production, gene therapies, and long-term pharmacology studies.

Expression in Plant Cells

Agrobacterium-mediated genetic transformation

(a) Depicts the process of disarming of the Agrobacteria tumor-inducing plasmid and fusion with plant viral regulatory sequences. (b) Depicts co-expression via infiltration of several Agrobacteria cultures containing separate binary vectors or cultures possessing single vectors containing multiple expression cassettes. Development-of-transient-expression-systems-for-use-in-plant-based-synthetic-biology-a.jpg
(a) Depicts the process of disarming of the Agrobacteria tumor-inducing plasmid and fusion with plant viral regulatory sequences. (b) Depicts co-expression via infiltration of several Agrobacteria cultures containing separate binary vectors or cultures possessing single vectors containing multiple expression cassettes.

The dominant technology used for the production of transgenic plants for transient expression is Agrobacterium-mediated genetic transformation, or "agroinfiltration," and virus expression machinery. [6] Agrobacterium tumefaciens and related Agrobacterium species are well-known plant pathogens that have been engineered to efficiently transfer specific pieces of DNA (called transfer DNA, or T-DNA) into the plant nucleus using binary vector systems, which consists of a T-DNA binary vector and a vir helper plasmid. [7] This binary vector separates T-DNA from trans-acting virulence proteins that help mediate the transfer. [8] Advantages of this method include modularity of broad host-range plasmids of small size through standard molecular biology techniques. Furthermore, since the parent tumor-inducing plasmid in Agrobacterium strains have been disarmed and only non-reproductive cells have been modified (as opposed to germ-line modifications), the process is considered environmentally harmless. [6]

Applications of this process has resulted in advancements made in the use of plants to synthetic biology. Plant-derived bioproducts show promise of high competitiveness towards traditional mammalian cell expression systems.

Expression in Mammalian Cells

Mammalian cell expression systems are essential for the transient production of recombinant proteins and their complementary post-translational modifications. In fact, approximately half of the current commercially available therapeutic proteins are produced in mammalian cells. However, mammalian cell systems' slow growth, precise growth requirements, and potential risk of infection by animal viruses present a number of challenges. As a result, a growing number of mammalian cell lines have been established to serve as hosts for transient recombinant protein production. [2]

HEK293 cells

Although other cells lines, such as African green monkey kidney (COS) and baby hamster kidney (BHK), can be used for recombinant protein production, the most commonly employed host system in transient expression of mammalian cells involves derivatives of the HEK293 cell line, which is based on the human embryonic kidney cell line established in 1977 by Graham et al. [9] The HEK293 cell line was created via transformation with sheared Adenovirus 5 DNA. [10] Advantages of using this cell line include their high rates of transfection and ability to grow in a serum-free medium, which results in reduced cost and lowered risk of contamination with animal-derived material typically found in serum. [2]

Several engineered sublines were later developed by incorporating viral elements derived from mammalian viruses, such as SV40 virus or Epstein–Barr virus (EBV), which are notable for their high retention of plasmid DNA in an episomal state and their capacity to increase transcription and translation via specific viral properties. [11] These later sublines were consequently identified to have two interacting components: the SV40 large T-antigen binding to the SV20 origin of replication (SV40ori) and the EBV-derived nuclear antigen-1 (EBNA-1) protein to its associated origin of replication (oriP). [11]

Typical historical yields of transient expression in HEK293 cells transfected using PEI-25kDa was 20-40 mg/L of recombinant antibody protein. In 2008, Backliwal et. al reported for the first time yields crossing 1 g/L of recombinant antibody protein. [12]

CHO cells

Traditionally, Chinese hamster ovary (CHO) cells are associated with the establishment of stable cell lines for biologics. Recently, however, attempts to engineer CHO cells for transient protein production have garnered recognition. CHO cells were among the earliest established cell lines for in vitro cultivation, and their potential as a host for production and manufacturing of biological products remains popular. [11] CHO cells are preferable for transient expression due to their easy industrial scale-up, versatility for the production of diverse biomolecules, and low risk of infection of human viruses, among other advantages. [13] Three primary expression systems have been established:

  1. EBNA-1-engineered CHO cell line
  2. CHO EBNA LT cell line, which is carried apart from the EBNA-1 gene and the mouse polyomavirus large T antigen [14]
  3. EpiCHO system, which consists of a CHO cell line transfected with the polyomavirus large T antigen gene and a DNA expression vector encoding polyomavirus origin (PyOri) for autonomous replication and EBV EBNA-1 and OriP for plasmid retention. [15] [16]

Related Research Articles

An episome is a special type of plasmid, which remains as a part of the eukaryotic genome without integration. Episomes manage this by replicating together with the rest of the genome and subsequently associating with metaphase chromosomes during mitosis. Episomes do not degrade, unlike standard plasmids, and can be designed so that they are not epigenetically silenced inside the eukaryotic cell nucleus. Episomes can be observed in nature in certain types of long-term infection by adeno-associated virus or Epstein-Barr virus. In 2004, it was proposed that non-viral episomes might be used in genetic therapy for long-term change in gene expression.

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids often carry useful genes, such as for antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

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

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.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

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.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

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.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

<span class="mw-page-title-main">Biotechnology in pharmaceutical manufacturing</span>

Biotechnology is the use of living organisms to develop useful products. Biotechnology is often used in pharmaceutical manufacturing. Notable examples include the use of bacteria to produce things such as insulin or human growth hormone. Other examples include the use of transgenic pigs for the creation of hemoglobin in use of humans.

<span class="mw-page-title-main">Agroinfiltration</span> Method in plant biotechnology

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.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

QMCF Technology is an episomal protein production system that uses genetically modified mammalian cells and specially designed plasmids. QMCF plasmids carry a combination of regulatory sequences from mouse polyomavirus (Py) DNA replication origin which in combination with Epstein-Barr virus (EBV) EBNA-1 protein binding site as nuclear retention elements ensure stable propagation of plasmids in mammalian cells. In addition the vectors carry the selection marker operational for selection of plasmid carrying bacteria and QMCF cells, bacterial ColE1 origin of replication, and cassette for expression of protein of interest. QMCF cell lines express Large-T antigen and EBNA-1 proteins which bind the viral sequences on the QMCF plasmid and hence support plasmid replication and maintenance in the cells. QMCF Technology has several important differences compared to commonly known transient expression and stable cell line expression systems. Unlike in transient expression system, QMCF Technology enables to maintain episomally replicating QMCF plasmids inside the cells for up to 50 days thus providing an option for production phase of 2–3 weeks. Therefore, the production levels of QMCF Technology are higher. Another difference is the option of establishing expression cell banks within one week, which is not feasible with transient system. Compared to usage of stable cell line, QMCF technology is a rapid method leaving out time-consuming clone selection step during cell line development.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">Vectors in gene therapy</span>

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

An edible vaccine is a food, typically plants, that contain vitamins, proteins or other nourishment that act as a vaccine against a certain disease. Once the plant, fruit, or plant derived product is ingested orally, it stimulates the immune system. Specifically, it stimulates both the mucosal and humoral immune systems. Edible vaccines are genetically modified crops that contain antigens for specific diseases. Edible vaccines offer many benefits over traditional vaccines, due to their lower manufacturing cost and a lack of negative side effects. However, there are limitations as edible vaccines are still new and developing. Further research will need to be done before they are ready for widespread human consumption. Edible vaccines are currently being developed for measles, cholera, foot and mouth disease, Hepatitis B and Hepatitis C.

<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. ↑ Hacker DL (2011-01-01). "2.29 - Recombinant Technology". In Moo-Young M, Wurm FM (eds.). Comprehensive Biotechnology (Second ed.). Burlington: Academic Press. pp. 401–406. doi:10.1016/b978-0-08-088504-9.00120-3. ISBN   978-0-08-088504-9.
  2. 1 2 3 Glick BR, Patten CL (January 2017). Molecular Biotechnology: Principles and Applications of Recombinant DNA (Fifth ed.). doi:10.1128/9781555819378. ISBN   9781555819378.
  3. ↑ Strohl WR, Strohl LM (2013). Therapeutic antibody engineering : current and future advances driving the strongest growth area in the pharmaceutical industry. Cambridge, UK: Woodhead Publishing. ISBN   978-1-907568-37-4. OCLC   870891982.
  4. ↑ Menassa R, Ahmad A, Joensuu JJ (2011-09-21). "Transient Expression Using Agroinfiltration and Its Applications in Molecular Farming". Molecular Farming in Plants: Recent Advances and Future Prospects. Dordrecht: Springer Netherlands. pp. 183–198. doi:10.1007/978-94-007-2217-0_9. ISBN   978-94-007-2216-3.
  5. ↑ Strohl WR, Strohl LM (2012). "Therapeutic antibody classes.". Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry (1st ed.). Sawston: Woodhead Publishing. pp. 197–223. doi:10.1533/9781908818096.197. ISBN   9781907568374.
  6. 1 2 Sainsbury F, Lomonossoff GP (June 2014). "Transient expressions of synthetic biology in plants". Current Opinion in Plant Biology. SI: Physiology and metabolism. 19 (100): 1–7. doi:10.1016/j.pbi.2014.02.003. PMC   4070481 . PMID   24631883.
  7. ↑ Bevan M (November 1984). "Binary Agrobacterium vectors for plant transformation". Nucleic Acids Research. 12 (22): 8711–8721. doi:10.1093/nar/12.22.8711. PMC   320409 . PMID   6095209.
  8. ↑ "Corrigendum to 'An Agrobacterium-mediated transient gene expression system for intact leaves' [Plant Sci. 122 (1997) 101–108]". Plant Science. 124 (2): 227. May 1997. doi: 10.1016/s0168-9452(97)04651-7 . ISSN   0168-9452.
  9. ↑ Voynov V, Caravella JA (2012). Therapeutic proteins : methods and protocols (2nd ed.). New York: Humana Press. ISBN   978-1-61779-921-1. OCLC   798095845.
  10. ↑ Graham FL, Smiley J, Russell WC, Nairn R (July 1977). "Characteristics of a human cell line transformed by DNA from human adenovirus type 5". The Journal of General Virology. 36 (1): 59–74. doi: 10.1099/0022-1317-36-1-59 . PMID   886304.
  11. 1 2 3 Geisse S, Voedisch B (2012). "Transient Expression Technologies: Past, Present, and Future". Therapeutic Proteins. Methods in Molecular Biology. Vol. 899. Totowa, NJ: Humana Press. pp. 203–219. doi:10.1007/978-1-61779-921-1_13. ISBN   978-1-61779-920-4. PMID   22735955.
  12. ↑ Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM (September 2008). "Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/L by transient transfection under serum-free conditions". Nucleic Acids Research. 36 (15): e96. doi:10.1093/nar/gkn423. PMC   2528171 . PMID   18617574.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. ↑ Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, Field R (2014). "A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression". Biotechnology Progress. 30 (1): 132–141. doi:10.1002/btpr.1809. PMID   24106171. S2CID   20068880.
  14. ↑ Silla T, Hääl I, Geimanen J, Janikson K, Abroi A, Ustav E, Ustav M (December 2005). "Episomal maintenance of plasmids with hybrid origins in mouse cells". Journal of Virology. 79 (24): 15277–15288. doi:10.1128/jvi.79.24.15277-15288.2005. PMC   1316011 . PMID   16306599.
  15. ↑ Codamo J, Munro TP, Hughes BS, Song M, Gray PP (June 2011). "Enhanced CHO cell-based transient gene expression with the epi-CHO expression system". Molecular Biotechnology. 48 (2): 109–115. doi:10.1007/s12033-010-9351-9. PMID   21104043. S2CID   42312966.
  16. ↑ Kunaparaju R, Liao M, Sunstrom NA (September 2005). "Epi-CHO, an episomal expression system for recombinant protein production in CHO cells". Biotechnology and Bioengineering. 91 (6): 670–677. doi: 10.1002/bit.20534 . hdl: 1959.4/41499 . PMID   15948170.
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