Transient expression

<h2 id="advantages">Advantages</h2>
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 <a href="/facts/RNA_interference/oJRWBXJH">RNA interference</a> (RNAi)‑mediated gene silencing, or quickly generate small-scale recombinant proteins.<a class="footnote-ref" id="fnref:3" href="#fn:3">3</a> 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.<a class="footnote-ref" id="fnref:4" href="#fn:4">4</a> 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 <a href="/facts/Translocation_(genetics)/MKEwqpQw">translocation</a> to the nucleus and <a href="/facts/Transcription_(biology)/bDgp9HDN">transcription</a>.<a class="footnote-ref" id="fnref:5" href="#fn:5">5</a>
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.

<h2 id="expression-in-plant-cells">Expression in Plant Cells</h2>
<h3>Agrobacterium-mediated genetic transformation</h3>

The dominant technology used for the production of transgenic plants for transient expression is Agrobacterium-mediated genetic transformation, or "agroinfiltration," and virus expression machinery.<a class="footnote-ref" id="fnref:6" href="#fn:6">6</a> 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 <a href="/facts/Transfer_DNA_binary_system/EE4zvpbn">binary vector systems</a>, which consists of a <a href="/facts/Transfer_DNA/8kQjXNWf">T-DNA</a> binary vector and a vir helper plasmid.<a class="footnote-ref" id="fnref:7" href="#fn:7">7</a> This binary vector separates T-DNA from trans-acting virulence proteins that help mediate the transfer.<a class="footnote-ref" id="fnref:8" href="#fn:8">8</a> 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.<a class="footnote-ref" id="fnref:9" href="#fn:9">9</a>
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.

<h2 id="expression-in-mammalian-cells">Expression in Mammalian Cells</h2>
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.<a class="footnote-ref" id="fnref:10" href="#fn:10">10</a>

<h3>HEK293 cells</h3>
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 <a href="/facts/HEK_293_cells/Xq6NaTq1">HEK293</a> cell line, which is based on the human embryonic kidney cell line established in 1977 by Graham et al.<a class="footnote-ref" id="fnref:11" href="#fn:11">11</a> The HEK293 cell line was created via transformation with sheared Adenovirus 5 DNA.<a class="footnote-ref" id="fnref:12" href="#fn:12">12</a> 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.<a class="footnote-ref" id="fnref:13" href="#fn:13">13</a>
Several engineered sublines were later developed by incorporating viral elements derived from mammalian viruses, such as SV40 virus or <a href="/facts/Epstein%E2%80%93Barr_virus/Uv8f1PNd">Epstein–Barr virus</a> (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.<a class="footnote-ref" id="fnref:14" href="#fn:14">14</a> 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 (<a href="/facts/EBNA-1/GuJHwIRl">EBNA-1</a>) protein to its associated origin of replication (oriP).<a class="footnote-ref" id="fnref:15" href="#fn:15">15</a>
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.<a class="footnote-ref" id="fnref:16" href="#fn:16">16</a>

<h3>CHO cells</h3>
Traditionally, <a href="/facts/Chinese_hamster_ovary_cell/t09KCNNc">Chinese hamster ovary (CHO) cells</a> 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.<a class="footnote-ref" id="fnref:17" href="#fn:17">17</a> 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.<a class="footnote-ref" id="fnref:18" href="#fn:18">18</a> Three primary expression systems have been established:

<ol><li>EBNA-1-engineered CHO cell line</li>
<li>CHO EBNA LT cell line, which is carried apart from the EBNA-1 gene and the mouse polyomavirus large T antigen<a class="footnote-ref" id="fnref:19" href="#fn:19">19</a></li>
<li>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.<a class="footnote-ref" id="fnref:20" href="#fn:20">20</a><a class="footnote-ref" id="fnref:21" href="#fn:21">21</a></li></ol>

<h2 id="external-links">External links</h2>
<ul><li>Canto T (2016). "Transient Expression Systems in Plants: Potentialities and Constraints". Advanced Technologies for Protein Complex Production and Characterization. Advances in Experimental Medicine and Biology. Vol. 896. pp. 287–301. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1007%2F978-3-319-27216-0_18">10.1007/978-3-319-27216-0_18</a>. <a href="/facts/Hdl_(identifier)/rdebSxmC">hdl</a>:<a href="https://hdl.handle.net/10261%2F135535">10261/135535</a>. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-3-319-27214-6. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/27165332">27165332</a>.</li>
<li>Grimsley N, Hohn B, Hohn T, Walden R (May 1986). <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC323497">""Agroinfection," an alternative route for viral infection of plants by using the Ti plasmid"</a>. Proceedings of the National Academy of Sciences of the United States of America. 83 (10): 3282–3286. <a href="/facts/Bibcode_(identifier)/9HtdQSGB">Bibcode</a>:<a href="https://ui.adsabs.harvard.edu/abs/1986PNAS...83.3282G">1986PNAS...83.3282G</a>. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1073%2Fpnas.83.10.3282">10.1073/pnas.83.10.3282</a>. <a href="/facts/PMC_(identifier)/dX1zMt71">PMC</a> <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC323497">323497</a>. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/16593697">16593697</a>.</li>
<li>Patrick M. <a href="https://blog.addgene.org/plasmids-101-yeast-vectors">"Plasmids 101: Yeast Vectors"</a>. blog.addgene.org.</li>
<li><a href="https://www.genscript.com/transient-vs-stable-expression.html">"Transient vs. Stable Expression"</a>. www.genscript.com.</li></ul>

<h2 id="references">References</h2>

<ol>
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<li id="fn:9">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. Bibcode:2014COPB...18D...5B. doi:10.1016/j.pbi.2014.02.003. PMC 4070481. PMID 24631883. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070481" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070481</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></li>
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</ol>

Transient expression open-in-new

Transient expression