Multicistronic expression systems enable production of multiple proteins from a single transcript, with internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides as common tools. Because 2A peptides rely on a single translation event, we tested whether nuclear-localized and secreted reporters separated by F2A in C. elegans produced the expected localization pattern. Both configurations showed efficient cleavage, with nuclear and secreted proteins localizing correctly, indicating that cleavage was effective and downstream secreted proteins could be correctly directed to the secretory pathway. These data provide a configuration for driving nuclear and secreted proteins from a single transgene.
","acknowledgements":"The authors thank Tabatha Wells for research support and Mike Nonet for plasmids. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). WormBase was used in the design and execution of experiments.
","authors":[{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["investigation","methodology","writing_reviewEditing"],"email":"jragle@ucsc.edu","firstName":"James Matthew","lastName":"Ragle","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"0000-0002-6626-2615"},{"affiliations":["University of California, Santa Cruz, Santa Cruz, California, United States"],"departments":["Department of Molecular, Cell, and Developmental Biology"],"credit":["investigation","writing_reviewEditing"],"email":"gashley@ucsc.edu","firstName":"Guinevere","lastName":"Ashley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3983-5553"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["formalAnalysis","fundingAcquisition","investigation","methodology","supervision","validation","visualization","writing_originalDraft"],"email":"jward2@ucsc.edu","firstName":"Jordan D.","lastName":"Ward","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9870-8936"}],"awards":[{"awardId":"R35GM158317","funderName":"National Institute of General Medical Sciences (United States)","awardRecipient":"Jordan D. Ward"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award R35GM158317 to J.D.W.
","image":{"url":"https://portal.micropublication.org/uploads/70a599fdcfb991721d319ac80e3afc46.png"},"imageCaption":"Dual reporter strains of the indicated genotype were imaged for mScarlet and mNeonGreen in a plane containing both coelomocytes and body wall muscle nuclei. A merged image overlaid on a DIC image is provided. Images are representative of 20 animals imaged over two independent experiments. Scale bars=10 µm.
","imageTitle":"An F2A sequence drives efficient cleavage and allows correct localization of nuclear and secreted protein reporters regardless of configuration.
","methods":"Cloning and strain generation
We generated F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR (pJW2791) and F2A::ssmScarlet (dpi)::ubl-1 3'UTR (pJW2792) plasmids (Twist Bioscience). These plasmids contained ATG and GTA connectors for SapTrap and a KpnI restriction enzyme site upstream of the F2A sequence to allow linearization to Gibson clone in new sequences. We amplified ssmScarlet from pJW2792 to Gibson clone into linearized pJW2791 to generate pJW2793 (F2A::ssmScarlet (dpi)::ubl-1 3'UTR ). Similarly, mStayGold::H2B was amplified from pJW2791 to Gibson clone into linearized pJW2792 to make pJW2794 (mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). ppJW2791-pJW2794 were combined with pNM4104 (myo-3p) into a rapid RMCE backbone (pNM4216) through SapTrap (Schwartz & Jorgensen, 2016) to generate pJW2826 (myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR),pJW2827 (myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR), pJW3015 (myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), and pJW3016 (myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2826, pJW2827, pJW3015, and pJW3016 were integrated into NM5548 using rapid RMCE as previously described (Nonet, 2023) to generate JDW972, JDW937, JDW1041, and JDW1042, respectively. Oligonucleotides and sequence files available upon request.
Imaging
L4 plus 1 day adults were mounted on glass slides in 24 ul M9 + 0.05% gelatin and 10mM levamisole and imaged at 100 ms (Alx488) and 200 ms (Alx549) using a Plan-Apochromat 100x/1.40 Oil M27 Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy, LLC) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera. Acquired images were processed through Zen 2.3 (blue edition).
","reagents":"Plasmid | Reference | Notes | How to obtain plasmid |
pJW2791 | This study | F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2792 | This study | F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2793 | This study | ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2794 | This study | mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW3015 | This study | myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW3016 | This study | myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pNM4104 | Gift from Mike Nonet | myo-3p clone with TGG and ATG connectors for SapTrap | Request from Mike Nonet |
pNM4216 (pHygG1) | Nonet, 2023 | Insertion backbone for rapid RMCE | Request from Mike Nonet |
pJW2826 | This study | myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW2827 | This study | myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
Strain | Genotype | Available from |
jsSi1726 [loxP myo-2p::FRT::nlsCyOFP::myo-2 3' + mex-5p::FLP D5::glh-2 3' FRT3] II | CGC | |
jsSi1579 jsSi1706 jsSi1726 wrdSi140[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1726 wrdSi149[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi175[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi176[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward |
Modern transgenesis frequently involves generating multiple proteins from a single mRNA under the control of a promoter of interest. Advantages of this approach include economical packaging in targeting vectors, avoiding the need for sequential genome manipulation, and creating reporters to track the expression of unmarked proteins of interest. These sequences can also be used in genome editing to create endogenous promoter reporters, capturing all relevant cis-regulatory elements. Three widely used approaches are internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides. Internal ribosome entry sites use viral sequences that allow cap-independent initiation of translation internally within an mRNA (Martinez-Salas et al., 2017). SL2 sequences exploit polycistronic operons found in some nematode species and involve splicing an SL2 leader RNA containing the 5' cap onto a downstream gene in the operon, producing two separate monocistronic mRNAs (Blumenthal, 2005; Spieth et al., 1993). 2A peptides are viral sequences that promote a ribosomal “skipping” event during translation that yields multiple, near-stoichiometric protein products from a single open reading frame (de Lima & Lanza, 2021). IRES, SL2, and 2A sequences can also be used to tag endogenous genes, allowing reporters or other proteins of interest to be expressed under the control of a gene of interest's cis-regulatory elements (Nance & Frøkjær-Jensen, 2019; Wang & Marchisio, 2021).
With IRES and SL2 sequences, separate translation initiation events produce the upstream and downstream proteins (Blumenthal, 2005; Martinez-Salas et al., 2017). In contrast, with 2A sequences, a single ribosome initiates translation and peptide cleavage produces separate polypeptides (de Lima & Lanza, 2021). This distinction raised the question as to whether targeting sequences on proteins such as nuclear localization sequences and signal peptides could function with 2A sequences. There are examples in which a cytosolic or nuclear-localized 2A::fluorescent reporter displayed correct localization downstream of a secreted protein (Rasala et al., 2012; Sun et al., 2023). However, there was a report where a downstream cytosolic 2A::reporter failed to cleave and was pulled into the secretory pathway through a proposed “slipstream” mechanism (de Felipe et al., 2010). The generalizability of this result is not clear, as another study found that a secreted protein downstream of a 2A sequence required its own signal sequence for secretion (Yan et al., 2010).
Given these variable reports and the wide use of 2A peptides in C. elegans transgenes (Ahier & Jarriault, 2014), we tested whether mStayGold::H2B and secreted mScarlet (signal sequence mScarlet; ssmScarlet) reporters separated by an F2A sequence expressed in body wall muscle displayed the expected localization pattern (Figure 1). In both configurations, we observed mScarlet signal accumulating in coelomocytes and mStayGold in muscle nuclei, suggesting efficient F2A cleavage. Downstream mStayGold::H2B did not enter the endoplasmic reticulum through a slipstream mechanism, and the ssmScarlet could efficiently enter the secretory pathway after 2A cleavage. We included F2A::H2B::mSG and F2A::ssmScarlet controls lacking the upstream FP, which also localized as expected. These results provide an effective design that allows efficient cleavage and correct localization of nuclear and secreted proteins separated by a 2A sequence. This design would likely support correct localization to other cellular compartments based on efficient 2A cleavage, though this assertion will need to be tested. This work indicates that expressing secreted proteins and proteins with specific sub-cellular localizations from single transgenes under promoters of interest is feasible, adding to the C. elegans toolkit.
","references":[{"reference":"Ahier A, Jarriault S. 2014. Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit. Genetics 196: 605-613.
","pubmedId":"","doi":"10.1534/genetics.113.160846"},{"reference":"Blumenthal T. 2005. Trans-splicing and operons. WormBook : 10.1895/wormbook.1.5.1.
","pubmedId":"","doi":"10.1895/wormbook.1.5.1"},{"reference":"de Felipe P, Luke GA, Brown JD, Ryan MD. 2010. Inhibition of 2A‐mediated ‘cleavage’ of certain artificial polyproteins bearing N‐terminal signal sequences. Biotechnology Journal 5: 213-223.
","pubmedId":"","doi":"10.1002/biot.200900134"},{"reference":"de Lima JGS, Lanza DCF. 2021. 2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology. Viruses 13: 2160.
","pubmedId":"","doi":"10.3390/v13112160"},{"reference":"Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Embarek AM. 2018. Insights into Structural and Mechanistic Features of Viral IRES Elements. Frontiers in Microbiology 8: 10.3389/fmicb.2017.02629.
","pubmedId":"","doi":"10.3389/fmicb.2017.02629"},{"reference":"Nance J, Frøkjær-Jensen C. 2019. The Caenorhabditis elegans Transgenic Toolbox. Genetics 212: 959-990.
","pubmedId":"","doi":"10.1534/genetics.119.301506"},{"reference":"Nonet ML. 2023. Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection. GENETICS 224: 10.1093/genetics/iyad072.
","pubmedId":"","doi":"10.1093/genetics/iyad072"},{"reference":"Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP. 2012. Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide. PLoS ONE 7: e43349.
","pubmedId":"","doi":"10.1371/journal.pone.0043349"},{"reference":"Schwartz ML, Jorgensen EM. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics 202: 1277-1288.
","pubmedId":"","doi":"10.1534/genetics.115.184275"},{"reference":"Spieth J, Brooke G, Kuersten S, Lea K, Blumenthal T. 1993. Operons in C. elegans: Polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-532.
","pubmedId":"","doi":"10.1016/0092-8674(93)90139-h"},{"reference":"Sun H, Beets I, Schafer W, Hobert O. 2023. Comparing engineered nuclear-localized reporter cassettes. MicroPubl Biol 2023: 10.17912/micropub.biology.001014.
","pubmedId":"38021170","doi":""},{"reference":"Wang X, Marchisio MA. 2021. Synthetic polycistronic sequences in eukaryotes. Synthetic and Systems Biotechnology 6: 254-261.
","pubmedId":"","doi":"10.1016/j.synbio.2021.09.003"},{"reference":"Yan J, Wang H, Xu Q, Jain N, Toxavidis V, Tigges J, et al., Gao. 2010. Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression. Analytical Biochemistry 399: 144-146.
","pubmedId":"","doi":"10.1016/j.ab.2009.11.032"}],"title":"An F2A sequence permits correct localization of a secreted and a nuclear localized reporter in C. elegans
","reviews":[{"reviewer":{"displayName":"Sophie Jarriault"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"43ec2e60-e32d-4d15-89f3-34890e4238ce","decision":"accept","abstract":"Multicistronic expression systems enable production of multiple proteins from a single transcript, with internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides as common tools. Because 2A peptides rely on a single translation event, we tested whether nuclear-localized mStayGold (mSG::H2B) and secreted mScarlet (ssmScarlet) reporters separated by F2A in C. elegans produced the expected localization pattern. mSG::H2B::F2A::ssmScarlet and ssmScarlet::F2A::mSG::H2B expressed in body wall muscle produced nuclear mSG in muscle and ssmScarlet in coelomocytes, indicating that downstream secreted proteins could be correctly directed to the secretory pathway. These data provide a configuration for driving nuclear and secreted proteins from a single-copy transgene.
","acknowledgements":"The authors thank Tabatha Wells for research support and Mike Nonet for plasmids. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). WormBase was used in the design and execution of experiments.
","authors":[{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["investigation","methodology","writing_reviewEditing"],"email":"jragle@ucsc.edu","firstName":"James Matthew","lastName":"Ragle","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"0000-0002-6626-2615"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":["Department of Molecular, Cell, and Developmental Biology"],"credit":["investigation","writing_reviewEditing"],"email":"gashley@ucsc.edu","firstName":"Guinevere","lastName":"Ashley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3983-5553"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["formalAnalysis","fundingAcquisition","investigation","methodology","supervision","validation","visualization","writing_originalDraft"],"email":"jward2@ucsc.edu","firstName":"Jordan D.","lastName":"Ward","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9870-8936"}],"awards":[{"awardId":"R35GM158317","funderName":"National Institute of General Medical Sciences (United States)","awardRecipient":"Jordan D. Ward"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award R35GM158317 to J.D.W.
","image":{"url":"https://portal.micropublication.org/uploads/d137431852ce13d77873092f9118ec29.png"},"imageCaption":"Dual reporter strains of the indicated genotype were imaged for secreted mScarlet (ssmScarlet) and mStayGold::histone H2B fusions (mSG::H2B). A merged image overlaid on a DIC image is provided and the outline of the coelomocyte cell body is indicated by a dashed yellow line in the DIC overlap images. Images are representative of 20 young adult animals imaged over two independent experiments. We selected images where coelomocyte and muscle nuclei could both be imaged in a single DIC plane and animal anterior is oriented to the left. The top two rows image the posterior pair of coelomocytes closest to the tail. The bottom two rows are of the mid-body pair of coelomocytes. Scale bars=10 µm.
","imageTitle":"An F2A sequence allows correct localization of nuclear and secreted protein reporters regardless of configuration
","methods":"Cloning and strain generation
F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR (pJW2791) and F2A::ssmScarlet (dpi)::ubl-1 3'UTR (pJW2792) plasmids were generated by Twist Biosciences, cloning the insert into a pTwist Kan High Copy backbone. The “dpi” designation refers to sequence optimization to remove piRNA target sites performed with the pirScan program (Wu et al., 2018). These plasmids contained ATG and GTA connectors for SapTrap and a KpnI restriction enzyme site upstream of the F2A sequence to allow linearization to Gibson clone in new sequences. We amplified ssmScarlet from pJW2792 to Gibson clone into linearized pJW2791 to generate pJW2793 (F2A::ssmScarlet (dpi)::ubl-1 3'UTR ). Similarly, mStayGold::H2B was amplified from pJW2791 to Gibson clone into linearized pJW2792 to make pJW2794 (mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2791-pJW2794 were combined with pNM4104 (myo-3p) into a rapid RMCE backbone (pNM4216) through SapTrap (Schwartz & Jorgensen, 2016) to generate pJW2826 (myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), pJW2827 (myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR), pJW3015 (myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), and pJW3016 (myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2826, pJW2827, pJW3015, and pJW3016 were integrated into NM5548 using rapid RMCE as previously described (Nonet, 2023) to generate JDW972, JDW937, JDW1041, and JDW1042, respectively. Oligonucleotides and sequence files available upon request.
Imaging
Day 1 adults were mounted on glass slides in 24 ul M9 + 0.05% gelatin and 10mM levamisole and imaged at 100 ms (Alx488) and 200 ms (Alx549) using a Plan-Apochromat 100x/1.40 Oil M27 Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy, LLC) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera. Acquired images were processed through Zen 2.3 (blue edition).
","reagents":"Plasmid | Reference | Notes | How to obtain plasmid |
pJW2791 | This study | F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2792 | This study | F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2793 | This study | ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2794 | This study | mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2826 | This study | myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW2827 | This study | myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW3015 | This study | myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW3016 | This study | myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pNM4104 | Gift from Mike Nonet | myo-3p clone with TGG and ATG connectors for SapTrap | Request from Mike Nonet |
pNM4216 (pHygG1) | Nonet, 2023 | Insertion backbone for rapid RMCE | Request from Mike Nonet |
Strain | Genotype | Available from |
jsSi1726 [loxP myo-2p::FRT::nlsCyOFP::myo-2 3' + mex-5p::FLP D5::glh-2 3' FRT3] II | CGC | |
jsSi1579 jsSi1706 jsSi1726 wrdSi140[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1726 wrdSi149[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi175[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi176[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward |
Modern transgenesis frequently involves generating multiple proteins from a single mRNA under the control of a promoter of interest. Advantages of this approach include economical packaging in targeting vectors, avoiding the need for sequential genome manipulation, and creating reporters to track the expression of unmarked proteins of interest. These sequences can also be used in genome editing to create endogenous promoter reporters, capturing all relevant cis-regulatory elements. Three widely used approaches are internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides. Internal ribosome entry sites use viral sequences that allow cap-independent initiation of translation internally within an mRNA (Martinez-Salas et al., 2017). SL2 sequences exploit polycistronic operons found in some nematode species and involve splicing an SL2 leader RNA containing the 5' cap onto a downstream gene in the operon, producing two separate monocistronic mRNAs (Blumenthal, 2005; Spieth et al., 1993). 2A peptides are viral sequences that promote a ribosomal “skipping” event during translation that yields multiple, near-stoichiometric protein products from a single open reading frame (de Lima & Lanza, 2021). IRES, SL2, and 2A sequences can also be used to tag endogenous genes, allowing reporters or other proteins of interest to be expressed under the control of a gene of interest's cis-regulatory elements (Nance & Frøkjær-Jensen, 2019; Wang & Marchisio, 2021).
With IRES and SL2 sequences, separate translation initiation events produce the upstream and downstream proteins (Blumenthal, 2005; Martinez-Salas et al., 2017). In contrast, with 2A sequences, a single ribosome initiates translation and peptide cleavage produces separate polypeptides (de Lima & Lanza, 2021). A proof-of-principle study established that several 2A peptides could be used in C. elegans to enable, from a single construct, efficient delivery of up to four proteins to distinct compartments such as the cytoplasm, nucleus, nuclear membrane or plasma membrane (Ahier & Jarriault, 2014). However, it remains unclear whether 2A sequences could simultaneously support targeting of proteins with nuclear localization signals and signal peptides. There are examples in which a cytosolic or nuclear-localized 2A::fluorescent reporter displayed correct localization downstream of a secreted protein (Rasala et al., 2012; Sun et al., 2023). However, there was a report where a downstream cytosolic 2A::reporter failed to cleave and was pulled into the secretory pathway through a proposed “slipstream” mechanism (de Felipe et al., 2010). The generalizability of this result is not clear, as another study found that a secreted protein downstream of a 2A sequence required its own signal sequence for secretion (Yan et al., 2010).
Given these variable reports and the wide use of 2A peptides in C. elegans transgenes (Ahier & Jarriault, 2014), we tested whether mStayGold::histone H2B (mSG::H2B) and secreted mScarlet (signal sequence mScarlet; ssmScarlet) reporters separated by an F2A sequence expressed in body wall muscle displayed the expected localization pattern (Figure 1). We chose F2A as we had used it extensively in our auxin-inducible degron work and it produced efficient cleavage (Ashley et al., 2021). Fluorescent proteins secreted from many tissues into the pseudocoelom are subsequently scavenged by coelomocytes and accumulate in these cells (Fares & Greenwald, 2001; Fitzgerald & Greenwald, 1995; Grant & Greenwald, 1997). GFP secreted from body wall muscle has been previously used to genetically dissect the endocytic pathway (Fares & Greenwald, 2001). In both configurations, we observed mScarlet signal accumulating in coelomocytes and mStayGold in muscle nuclei. Downstream mStayGold::H2B did not detectably enter the endoplasmic reticulum through a slipstream mechanism, and the ssmScarlet did appear to efficiently enter the secretory pathway after 2A cleavage. We included F2A:::mSG::H2B and F2A::ssmScarlet controls lacking the upstream FP, which also localized as expected. These results provide an effective design that allows correct localization of nuclear and secreted proteins separated by an F2A sequence. This design would likely support correct localization to other cellular compartments based on efficient 2A cleavage, though this assertion will need to be tested. We note that in this study cleavage was inferred by reporter localization, and in the future western blotting experiments would be valuable to directly assess cleavage efficiency and other 2A peptides should be similarly tested. A recent study using a similar recombinase-mediated single-copy integration approach in CHO cells displayed incomplete cleavage (Ng et al., 2025). In contrast, our study supports the robust function of F2A peptides in single-copy transgenes inserted by rapid recombinase mediated cassette exchange in C. elegans. Together, this work indicates that expressing secreted proteins and proteins with specific sub-cellular localizations from single transgenes under promoters of interest is feasible, adding to the C. elegans toolkit.
","references":[{"reference":"Ahier A, Jarriault S. 2014. Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit. Genetics 196: 605-613.
","pubmedId":"","doi":"10.1534/genetics.113.160846"},{"reference":"Blumenthal T. 2005. Trans-splicing and operons. WormBook : 10.1895/wormbook.1.5.1.
","pubmedId":"","doi":"10.1895/wormbook.1.5.1"},{"reference":"de Felipe P, Luke GA, Brown JD, Ryan MD. 2010. Inhibition of 2A‐mediated ‘cleavage’ of certain artificial polyproteins bearing N‐terminal signal sequences. Biotechnology Journal 5: 213-223.
","pubmedId":"","doi":"10.1002/biot.200900134"},{"reference":"de Lima JGS, Lanza DCF. 2021. 2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology. Viruses 13: 2160.
","pubmedId":"","doi":"10.3390/v13112160"},{"reference":"Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Embarek AM. 2018. Insights into Structural and Mechanistic Features of Viral IRES Elements. Frontiers in Microbiology 8: 10.3389/fmicb.2017.02629.
","pubmedId":"","doi":"10.3389/fmicb.2017.02629"},{"reference":"Nance J, Frøkjær-Jensen C. 2019. The Caenorhabditis elegans Transgenic Toolbox. Genetics 212: 959-990.
","pubmedId":"","doi":"10.1534/genetics.119.301506"},{"reference":"Nonet ML. 2023. Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection. GENETICS 224: 10.1093/genetics/iyad072.
","pubmedId":"","doi":"10.1093/genetics/iyad072"},{"reference":"Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP. 2012. Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide. PLoS ONE 7: e43349.
","pubmedId":"","doi":"10.1371/journal.pone.0043349"},{"reference":"Schwartz ML, Jorgensen EM. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics 202: 1277-1288.
","pubmedId":"","doi":"10.1534/genetics.115.184275"},{"reference":"Spieth J, Brooke G, Kuersten S, Lea K, Blumenthal T. 1993. Operons in C. elegans: Polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-532.
","pubmedId":"","doi":"10.1016/0092-8674(93)90139-h"},{"reference":"Sun H, Beets I, Schafer W, Hobert O. 2023. Comparing engineered nuclear-localized reporter cassettes. MicroPubl Biol 2023: 10.17912/micropub.biology.001014.
","pubmedId":"38021170","doi":""},{"reference":"Wang X, Marchisio MA. 2021. Synthetic polycistronic sequences in eukaryotes. Synthetic and Systems Biotechnology 6: 254-261.
","pubmedId":"","doi":"10.1016/j.synbio.2021.09.003"},{"reference":"Yan J, Wang H, Xu Q, Jain N, Toxavidis V, Tigges J, et al., Gao. 2010. Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression. Analytical Biochemistry 399: 144-146.
","pubmedId":"","doi":"10.1016/j.ab.2009.11.032"},{"reference":"Fares H, Greenwald I. 2001. Genetic Analysis of Endocytosis in Caenorhabditis elegans: Coelomocyte Uptake Defective Mutants. Genetics 159: 133-145.
","pubmedId":"","doi":"10.1093/genetics/159.1.133"},{"reference":"Fitzgerald K, Greenwald I. 1995. Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo. Development 121: 4275-4282.
","pubmedId":"","doi":"10.1242/dev.121.12.4275"},{"reference":"Grant B, Greenwald I. 1997. Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans. Development 124: 637-644.
","pubmedId":"","doi":"10.1242/dev.124.3.637"},{"reference":"Ashley GE, Duong T, Levenson MT, Martinez MAQ, Johnson LC, Hibshman JD, et al., Ward. 2021. An expanded auxin-inducible degron toolkit for\n Caenorhabditis elegans. Genetics 217: 10.1093/genetics/iyab006.
","pubmedId":"","doi":"10.1093/genetics/iyab006"},{"reference":"Wu WS, Huang WC, Brown JS, Zhang D, Song X, Chen H, et al., Lee. 2018. pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans. Nucleic Acids Research 46: W43-W48.
","pubmedId":"","doi":"10.1093/nar/gky277"},{"reference":"Ng JPZ, Mariati M, Bi J, Chang MW, Yang Y. 2025. A Targeted Integration-Based CHO Cell Platform for Simultaneous Antibody Display and Secretion. Antibodies 14: 38.
","pubmedId":"","doi":"10.3390/antib14020038"}],"title":"An F2A sequence permits correct localization of a secreted and a nuclear localized reporter in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1788896847324"}]},{"id":"f28a97dd-bc29-41df-a263-af71075c000f","decision":"accept","abstract":"Multicistronic expression systems enable production of multiple proteins from a single transcript, with internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides as common tools. Because 2A peptides rely on a single translation event, we tested whether nuclear-localized mStayGold (mSG::H2B) and secreted mScarlet (ssmScarlet) reporters separated by F2A in C. elegans produced the expected localization pattern. mSG::H2B::F2A::ssmScarlet and ssmScarlet::F2A::mSG::H2B expressed in body wall muscle produced nuclear mSG in muscle and ssmScarlet in coelomocytes, indicating that downstream secreted proteins could be correctly directed to the secretory pathway. These data provide a configuration for driving nuclear and secreted proteins from a single-copy transgene.
","acknowledgements":"The authors thank Tabatha Wells for research support and Mike Nonet for plasmids. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). WormBase was used in the design and execution of experiments.
","authors":[{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["investigation","methodology","writing_reviewEditing"],"email":"jragle@ucsc.edu","firstName":"James Matthew","lastName":"Ragle","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"0000-0002-6626-2615"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["investigation","writing_reviewEditing"],"email":"gashley@ucsc.edu","firstName":"Guinevere","lastName":"Ashley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3983-5553"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064"],"departments":[""],"credit":["formalAnalysis","fundingAcquisition","investigation","methodology","supervision","validation","visualization","writing_originalDraft"],"email":"jward2@ucsc.edu","firstName":"Jordan D.","lastName":"Ward","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9870-8936"}],"awards":[{"awardId":"R35GM158317","funderName":"National Institute of General Medical Sciences (United States)","awardRecipient":"Jordan D. Ward"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award R35GM158317 to J.D.W.
","image":{"url":"https://portal.micropublication.org/uploads/d137431852ce13d77873092f9118ec29.png"},"imageCaption":"Dual reporter strains of the indicated genotype were imaged for secreted mScarlet (ssmScarlet) and mStayGold::histone H2B fusions (mSG::H2B). A merged image overlaid on a DIC image is provided and the outline of the coelomocyte cell body is indicated by a dashed yellow line in the DIC overlap images. Images are representative of 20 young adult animals imaged over two independent experiments. We selected images where coelomocyte and muscle nuclei could both be imaged in a single DIC plane and animal anterior is oriented to the left. The top row contains images of the posterior pair of coelomocytes closest to the tail. The bottom three rows contain images of the mid-body pair of coelomocytes. Scale bars=10 µm.
","imageTitle":"An F2A sequence allows correct localization of nuclear and secreted protein reporters regardless of configuration
","methods":"Cloning and strain generation
F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR (pJW2791) and F2A::ssmScarlet (dpi)::ubl-1 3'UTR (pJW2792) plasmids were generated by Twist Biosciences, cloning the insert into a pTwist Kan High Copy backbone. The “dpi” designation refers to sequence optimization to remove piRNA target sites performed with the pirScan program (Wu et al., 2018). These plasmids contained ATG and GTA connectors for SapTrap and a KpnI restriction enzyme site upstream of the F2A sequence to allow linearization to Gibson clone in new sequences. We amplified ssmScarlet from pJW2792 to Gibson clone into linearized pJW2791 to generate pJW2793 (F2A::ssmScarlet (dpi)::ubl-1 3'UTR ). Similarly, mStayGold::H2B was amplified from pJW2791 to Gibson clone into linearized pJW2792 to make pJW2794 (mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2791-pJW2794 were combined with pNM4104 (myo-3p) into a rapid RMCE backbone (pNM4216) through SapTrap (Schwartz & Jorgensen, 2016) to generate pJW2826 (myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), pJW2827 (myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR), pJW3015 (myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), and pJW3016 (myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2826, pJW2827, pJW3015, and pJW3016 were integrated into NM5548 using rapid RMCE as previously described (Nonet, 2023) to generate JDW972, JDW937, JDW1041, and JDW1042, respectively. Oligonucleotides and sequence files available upon request.
Imaging
Day 1 adults were mounted on glass slides in 24 ul M9 + 0.05% gelatin and 10mM levamisole and imaged at 100 ms (Alx488) and 200 ms (Alx549) using a Plan-Apochromat 100x/1.40 Oil M27 Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy, LLC) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera. Acquired images were processed through Zen 2.3 (blue edition).
","reagents":"Plasmid | Reference | Notes | How to obtain plasmid |
pJW2791 | This study | F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2792 | This study | F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2793 | This study | ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2794 | This study | mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2826 | This study | myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW2827 | This study | myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW3015 | This study | myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW3016 | This study | myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pNM4104 | Gift from Mike Nonet | myo-3p clone with TGG and ATG connectors for SapTrap | Request from Mike Nonet |
pNM4216 (pHygG1) | Nonet, 2023 | Insertion backbone for rapid RMCE | Request from Mike Nonet |
Strain | Genotype | Available from |
jsSi1726 [loxP myo-2p::FRT::nlsCyOFP::myo-2 3' + mex-5p::FLP D5::glh-2 3' FRT3] II | CGC | |
jsSi1579 jsSi1706 jsSi1726 wrdSi140[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1726 wrdSi149[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi175[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi176[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward |
Modern transgenesis frequently involves generating multiple proteins from a single mRNA under the control of a promoter of interest. Advantages of this approach include economical packaging in targeting vectors, avoiding the need for sequential genome manipulation, and creating reporters to track the expression of unmarked proteins of interest. These sequences can also be used in genome editing to create endogenous promoter reporters, capturing all relevant cis-regulatory elements. Three widely used approaches are internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides. Internal ribosome entry sites use viral sequences that allow cap-independent initiation of translation internally within an mRNA (Martinez-Salas et al., 2017). SL2 sequences exploit polycistronic operons found in some nematode species and involve splicing an SL2 leader RNA containing the 5' cap onto a downstream gene in the operon, producing two separate monocistronic mRNAs (Blumenthal, 2005; Spieth et al., 1993). 2A peptides are viral sequences that promote a ribosomal “skipping” event during translation that yields multiple, near-stoichiometric protein products from a single open reading frame (de Lima & Lanza, 2021). IRES, SL2, and 2A sequences can also be used to tag endogenous genes, allowing reporters or other proteins of interest to be expressed under the control of a gene of interest's cis-regulatory elements (Nance & Frøkjær-Jensen, 2019; Wang & Marchisio, 2021).
With IRES and SL2 sequences, separate translation initiation events produce the upstream and downstream proteins (Blumenthal, 2005; Martinez-Salas et al., 2017). In contrast, with 2A sequences, a single ribosome initiates translation and peptide cleavage produces separate polypeptides (de Lima & Lanza, 2021). A proof-of-principle study established that several 2A peptides could be used in C. elegans to enable, from a single construct, efficient delivery of up to four proteins to distinct compartments such as the cytoplasm, nucleus, nuclear membrane or plasma membrane (Ahier & Jarriault, 2014). However, it remains unclear whether 2A sequences could simultaneously support targeting of proteins with nuclear localization signals and signal peptides. There are examples in which a cytosolic or nuclear-localized 2A::fluorescent reporter displayed correct localization downstream of a secreted protein (Rasala et al., 2012; Sun et al., 2023). However, there was a report where a downstream cytosolic 2A::reporter failed to cleave and was pulled into the secretory pathway through a proposed “slipstream” mechanism (de Felipe et al., 2010). The generalizability of this result is not clear, as another study found that a secreted protein downstream of a 2A sequence required its own signal sequence for secretion (Yan et al., 2010).
Given these variable reports and the wide use of 2A peptides in C. elegans transgenes (Ahier & Jarriault, 2014), we tested whether mStayGold::histone H2B (mSG::H2B) and secreted mScarlet (signal sequence mScarlet; ssmScarlet) reporters separated by an F2A sequence expressed in body wall muscle displayed the expected localization pattern (Figure 1). We chose F2A as we had used it extensively in our auxin-inducible degron work and it produced efficient cleavage (Ashley et al., 2021). Fluorescent proteins secreted from many tissues into the pseudocoelom are subsequently scavenged by coelomocytes and accumulate in these cells (Fares & Greenwald, 2001; Fitzgerald & Greenwald, 1995; Grant & Greenwald, 1997). GFP secreted from body wall muscle has been previously used to genetically dissect the endocytic pathway (Fares & Greenwald, 2001). In both configurations, we observed mScarlet signal accumulating in coelomocytes and mStayGold in muscle nuclei. Downstream mStayGold::H2B did not detectably enter the endoplasmic reticulum through a slipstream mechanism, and the ssmScarlet did appear to efficiently enter the secretory pathway after 2A cleavage. We included F2A::mSG::H2B and F2A::ssmScarlet controls lacking the upstream FP, which also localized as expected. These results provide an effective design that allows correct localization of nuclear and secreted proteins separated by an F2A sequence. This design would likely support correct localization to other cellular compartments based on efficient 2A cleavage, though this assertion will need to be tested. We note that in this study cleavage was inferred by reporter localization, and in the future western blotting experiments would be valuable to directly assess cleavage efficiency and other 2A peptides should be similarly tested. A recent study using a similar recombinase-mediated single-copy integration approach in CHO cells displayed incomplete cleavage (Ng et al., 2025). In contrast, our study supports the robust function of F2A peptides in single-copy transgenes inserted by rapid recombinase mediated cassette exchange in C. elegans. Together, this work indicates that expressing secreted proteins and proteins with specific sub-cellular localizations from single transgenes under promoters of interest is feasible, adding to the C. elegans toolkit.
","references":[{"reference":"Ahier A, Jarriault S. 2014. Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit. Genetics 196: 605-613.
","pubmedId":"","doi":"10.1534/genetics.113.160846"},{"reference":"Ashley GE, Duong T, Levenson MT, Martinez MAQ, Johnson LC, Hibshman JD, et al., Ward. 2021. An expanded auxin-inducible degron toolkit for\n Caenorhabditis elegans. Genetics 217: 10.1093/genetics/iyab006.
","pubmedId":"","doi":"10.1093/genetics/iyab006"},{"reference":"Blumenthal T. 2005. Trans-splicing and operons. WormBook : 10.1895/wormbook.1.5.1.
","pubmedId":"","doi":"10.1895/wormbook.1.5.1"},{"reference":"de Felipe P, Luke GA, Brown JD, Ryan MD. 2010. Inhibition of 2A‐mediated ‘cleavage’ of certain artificial polyproteins bearing N‐terminal signal sequences. Biotechnology Journal 5: 213-223.
","pubmedId":"","doi":"10.1002/biot.200900134"},{"reference":"de Lima JGS, Lanza DCF. 2021. 2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology. Viruses 13: 2160.
","pubmedId":"","doi":"10.3390/v13112160"},{"reference":"Fares H, Greenwald I. 2001. Genetic Analysis of Endocytosis in Caenorhabditis elegans: Coelomocyte Uptake Defective Mutants. Genetics 159: 133-145.
","pubmedId":"","doi":"10.1093/genetics/159.1.133"},{"reference":"Fitzgerald K, Greenwald I. 1995. Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo. Development 121: 4275-4282.
","pubmedId":"","doi":"10.1242/dev.121.12.4275"},{"reference":"Grant B, Greenwald I. 1997. Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans. Development 124: 637-644.
","pubmedId":"","doi":"10.1242/dev.124.3.637"},{"reference":"Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Embarek AM. 2018. Insights into Structural and Mechanistic Features of Viral IRES Elements. Frontiers in Microbiology 8: 10.3389/fmicb.2017.02629.
","pubmedId":"","doi":"10.3389/fmicb.2017.02629"},{"reference":"Nance J, Frøkjær-Jensen C. 2019. The Caenorhabditis elegans Transgenic Toolbox. Genetics 212: 959-990.
","pubmedId":"","doi":"10.1534/genetics.119.301506"},{"reference":"Ng JPZ, Mariati M, Bi J, Chang MW, Yang Y. 2025. A Targeted Integration-Based CHO Cell Platform for Simultaneous Antibody Display and Secretion. Antibodies 14: 38.
","pubmedId":"","doi":"10.3390/antib14020038"},{"reference":"Nonet ML. 2023. Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection. GENETICS 224: 10.1093/genetics/iyad072.
","pubmedId":"","doi":"10.1093/genetics/iyad072"},{"reference":"Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP. 2012. Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide. PLoS ONE 7: e43349.
","pubmedId":"","doi":"10.1371/journal.pone.0043349"},{"reference":"Schwartz ML, Jorgensen EM. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics 202: 1277-1288.
","pubmedId":"","doi":"10.1534/genetics.115.184275"},{"reference":"Spieth J, Brooke G, Kuersten S, Lea K, Blumenthal T. 1993. Operons in C. elegans: Polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-532.
","pubmedId":"","doi":"10.1016/0092-8674(93)90139-h"},{"reference":"Sun H, Beets I, Schafer W, Hobert O. 2023. Comparing engineered nuclear-localized reporter cassettes. MicroPubl Biol 2023: 10.17912/micropub.biology.001014.
","pubmedId":"38021170","doi":""},{"reference":"Wang X, Marchisio MA. 2021. Synthetic polycistronic sequences in eukaryotes. Synthetic and Systems Biotechnology 6: 254-261.
","pubmedId":"","doi":"10.1016/j.synbio.2021.09.003"},{"reference":"Wu WS, Huang WC, Brown JS, Zhang D, Song X, Chen H, et al., Lee. 2018. pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans. Nucleic Acids Research 46: W43-W48.
","pubmedId":"","doi":"10.1093/nar/gky277"},{"reference":"Yan J, Wang H, Xu Q, Jain N, Toxavidis V, Tigges J, et al., Gao. 2010. Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression. Analytical Biochemistry 399: 144-146.
","pubmedId":"","doi":"10.1016/j.ab.2009.11.032"}],"title":"An F2A sequence permits correct localization of a secreted and a nuclear localized reporter in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"b46b94f1-9e58-42f2-86f9-cce1ebdb789b","decision":"publish","abstract":"Multicistronic expression systems enable production of multiple proteins from a single transcript, with internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides as common tools. Because 2A peptides rely on a single translation event, we tested whether nuclear-localized mStayGold (mSG::H2B) and secreted mScarlet (ssmScarlet) reporters separated by F2A in C. elegans produced the expected localization pattern. mSG::H2B::F2A::ssmScarlet and ssmScarlet::F2A::mSG::H2B expressed in body wall muscle produced nuclear mSG in muscle and ssmScarlet in coelomocytes, indicating that downstream secreted proteins could be correctly directed to the secretory pathway. These data provide a configuration for driving nuclear and secreted proteins from a single-copy transgene.
","acknowledgements":"The authors thank Tabatha Wells for research support and Mike Nonet for plasmids. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). WormBase was used in the design and execution of experiments.
","authors":[{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064, USA"],"departments":[""],"credit":["investigation","methodology","writing_reviewEditing"],"email":"jragle@ucsc.edu","firstName":"James Matthew","lastName":"Ragle","submittingAuthor":false,"correspondingAuthor":null,"equalContribution":null,"WBId":"","orcid":"0000-0002-6626-2615"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064, USA"],"departments":[""],"credit":["investigation","writing_reviewEditing"],"email":"gashley@ucsc.edu","firstName":"Guinevere","lastName":"Ashley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3983-5553"},{"affiliations":["Department of Molecular, Cell, and Developmental Biology, University of California – Santa Cruz, Santa Cruz, CA 95064, USA"],"departments":[""],"credit":["formalAnalysis","fundingAcquisition","investigation","methodology","supervision","validation","visualization","writing_originalDraft"],"email":"jward2@ucsc.edu","firstName":"Jordan D.","lastName":"Ward","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":"","orcid":"0000-0001-9870-8936"}],"awards":[{"awardId":"R35GM158317","funderName":"National Institute of General Medical Sciences (United States)","awardRecipient":"Jordan D. Ward"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award R35GM158317 to J.D.W.
","image":{"url":"https://portal.micropublication.org/uploads/d137431852ce13d77873092f9118ec29.png"},"imageCaption":"Dual reporter strains of the indicated genotype were imaged for secreted mScarlet (ssmScarlet) and mStayGold::histone H2B fusions (mSG::H2B). A merged image overlaid on a DIC image is provided and the outline of the coelomocyte cell body is indicated by a dashed yellow line in the DIC overlap images. Images are representative of 20 young adult animals imaged over two independent experiments. We selected images where coelomocyte and muscle nuclei could both be imaged in a single DIC plane and animal anterior is oriented to the left. The top row contains images of the posterior pair of coelomocytes closest to the tail. The bottom three rows contain images of the mid-body pair of coelomocytes. Scale bars=10 µm.
","imageTitle":"An F2A sequence allows correct localization of nuclear and secreted protein reporters regardless of configuration
","methods":"Cloning and strain generation
F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR (pJW2791) and F2A::ssmScarlet (dpi)::ubl-1 3'UTR (pJW2792) plasmids were generated by Twist Biosciences, cloning the insert into a pTwist Kan High Copy backbone. The “dpi” designation refers to sequence optimization to remove piRNA target sites performed with the pirScan program (Wu et al., 2018). These plasmids contained ATG and GTA connectors for SapTrap and a KpnI restriction enzyme site upstream of the F2A sequence to allow linearization to Gibson clone in new sequences. We amplified ssmScarlet from pJW2792 to Gibson clone into linearized pJW2791 to generate pJW2793 (F2A::ssmScarlet (dpi)::ubl-1 3'UTR ). Similarly, mStayGold::H2B was amplified from pJW2791 to Gibson clone into linearized pJW2792 to make pJW2794 (mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2791-pJW2794 were combined with pNM4104 (myo-3p) into a rapid RMCE backbone (pNM4216) through SapTrap (Schwartz & Jorgensen, 2016) to generate pJW2826 (myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), pJW2827 (myo-3p::mStayGold (dpi)::H2B::F2A:: ssmScarlet (dpi)::ubl-1 3'UTR), pJW3015 (myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR), and pJW3016 (myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR). pJW2826, pJW2827, pJW3015, and pJW3016 were integrated into NM5548 using rapid RMCE as previously described (Nonet, 2023) to generate JDW972, JDW937, JDW1041, and JDW1042, respectively. Oligonucleotides and sequence files available upon request.
Imaging
Day 1 adults were mounted on glass slides in 24 ul M9 + 0.05% gelatin and 10mM levamisole and imaged at 100 ms (Alx488) and 200 ms (Alx549) using a Plan-Apochromat 100x/1.40 Oil M27 Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy, LLC) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera. Acquired images were processed through Zen 2.3 (blue edition).
","reagents":"Plasmid | Reference | Notes | How to obtain plasmid |
pJW2791 | This study | F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2792 | This study | F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors. Has a KpnI site to clone in ssmScarlet or other factors in front of F2A | Request from Jordan Ward |
pJW2793 | This study | ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2794 | This study | mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW2826 | This study | myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW2827 | This study | myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR vector for rapid RMCE | Request from Jordan Ward |
pJW3015 | This study | myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pJW3016 | This study | myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR for SapTrap with ATG and GTA connectors | Request from Jordan Ward |
pNM4104 | Gift from Mike Nonet | myo-3p clone with TGG and ATG connectors for SapTrap | Request from Mike Nonet |
pNM4216 (pHygG1) | Nonet, 2023 | Insertion backbone for rapid RMCE | Request from Mike Nonet |
Strain | Genotype | Available from |
jsSi1726 [loxP myo-2p::FRT::nlsCyOFP::myo-2 3' + mex-5p::FLP D5::glh-2 3' FRT3] II | CGC | |
jsSi1579 jsSi1706 jsSi1726 wrdSi140[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::mStayGold (dpi)::H2B::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1726 wrdSi149[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::ssmScarlet (dpi)::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi175[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::mStayGold (dpi)::H2B::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward | |
jsSi1579 jsSi1706 jsSi1726 wrdSi176[loxP myo-2p::NLS::mNeonGreen, rps-0p HygR, loxP myo-3p::F2A::ssmScarlet (dpi)::ubl-1 3'UTR FRT3] II | Prof. Jordan Ward |
Modern transgenesis frequently involves generating multiple proteins from a single mRNA under the control of a promoter of interest. Advantages of this approach include economical packaging in targeting vectors, avoiding the need for sequential genome manipulation, and creating reporters to track the expression of unmarked proteins of interest. These sequences can also be used in genome editing to create endogenous promoter reporters, capturing all relevant cis-regulatory elements. Three widely used approaches are internal ribosome entry sites (IRES), SL2 trans-splicing, and 2A peptides. Internal ribosome entry sites use viral sequences that allow cap-independent initiation of translation internally within an mRNA (Martinez-Salas et al., 2017). SL2 sequences exploit polycistronic operons found in some nematode species and involve splicing an SL2 leader RNA containing the 5' cap onto a downstream gene in the operon, producing two separate monocistronic mRNAs (Blumenthal, 2005; Spieth et al., 1993). 2A peptides are viral sequences that promote a ribosomal “skipping” event during translation that yields multiple, near-stoichiometric protein products from a single open reading frame (de Lima & Lanza, 2021). IRES, SL2, and 2A sequences can also be used to tag endogenous genes, allowing reporters or other proteins of interest to be expressed under the control of a gene of interest's cis-regulatory elements (Nance & Frøkjær-Jensen, 2019; Wang & Marchisio, 2021).
With IRES and SL2 sequences, separate translation initiation events produce the upstream and downstream proteins (Blumenthal, 2005; Martinez-Salas et al., 2017). In contrast, with 2A sequences, a single ribosome initiates translation and peptide cleavage produces separate polypeptides (de Lima & Lanza, 2021). A proof-of-principle study established that several 2A peptides could be used in C. elegans to enable, from a single construct, efficient delivery of up to four proteins to distinct compartments such as the cytoplasm, nucleus, nuclear membrane or plasma membrane (Ahier & Jarriault, 2014). However, it remains unclear whether 2A sequences could simultaneously support targeting of proteins with nuclear localization signals and signal peptides. There are examples in which a cytosolic or nuclear-localized 2A::fluorescent reporter displayed correct localization downstream of a secreted protein (Rasala et al., 2012; Sun et al., 2023). However, there was a report where a downstream cytosolic 2A::reporter failed to cleave and was pulled into the secretory pathway through a proposed “slipstream” mechanism (de Felipe et al., 2010). The generalizability of this result is not clear, as another study found that a secreted protein downstream of a 2A sequence required its own signal sequence for secretion (Yan et al., 2010).
Given these variable reports and the wide use of 2A peptides in C. elegans transgenes (Ahier & Jarriault, 2014), we tested whether mStayGold::histone H2B (mSG::H2B) and secreted mScarlet (signal sequence mScarlet; ssmScarlet) reporters separated by an F2A sequence expressed in body wall muscle displayed the expected localization pattern (Figure 1). We chose F2A as we had used it extensively in our auxin-inducible degron work and it produced efficient cleavage (Ashley et al., 2021). Fluorescent proteins secreted from many tissues into the pseudocoelom are subsequently scavenged by coelomocytes and accumulate in these cells (Fares & Greenwald, 2001; Fitzgerald & Greenwald, 1995; Grant & Greenwald, 1997). GFP secreted from body wall muscle has been previously used to genetically dissect the endocytic pathway (Fares & Greenwald, 2001). In both configurations, we observed mScarlet signal accumulating in coelomocytes and mStayGold in muscle nuclei. Downstream mStayGold::H2B did not detectably enter the endoplasmic reticulum through a slipstream mechanism, and the ssmScarlet did appear to efficiently enter the secretory pathway after 2A cleavage. We included F2A::mSG::H2B and F2A::ssmScarlet controls lacking the upstream FP, which also localized as expected. These results provide an effective design that allows correct localization of nuclear and secreted proteins separated by an F2A sequence. This design would likely support correct localization to other cellular compartments based on efficient 2A cleavage, though this assertion will need to be tested. We note that in this study cleavage was inferred by reporter localization, and in the future western blotting experiments would be valuable to directly assess cleavage efficiency and other 2A peptides should be similarly tested. A recent study using a similar recombinase-mediated single-copy integration approach in CHO cells displayed incomplete cleavage (Ng et al., 2025). In contrast, our study supports the robust function of F2A peptides in single-copy transgenes inserted by rapid recombinase mediated cassette exchange in C. elegans. Together, this work indicates that expressing secreted proteins and proteins with specific sub-cellular localizations from single transgenes under promoters of interest is feasible, adding to the C. elegans toolkit.
","references":[{"reference":"Ahier A, Jarriault S. 2014. Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit. Genetics 196: 605-613.
","pubmedId":"","doi":"10.1534/genetics.113.160846"},{"reference":"Ashley GE, Duong T, Levenson MT, Martinez MAQ, Johnson LC, Hibshman JD, et al., Ward. 2021. An expanded auxin-inducible degron toolkit for\n Caenorhabditis elegans. Genetics 217: 10.1093/genetics/iyab006.
","pubmedId":"","doi":"10.1093/genetics/iyab006"},{"reference":"Blumenthal T. 2005. Trans-splicing and operons. WormBook : 10.1895/wormbook.1.5.1.
","pubmedId":"","doi":"10.1895/wormbook.1.5.1"},{"reference":"de Felipe P, Luke GA, Brown JD, Ryan MD. 2010. Inhibition of 2A‐mediated ‘cleavage’ of certain artificial polyproteins bearing N‐terminal signal sequences. Biotechnology Journal 5: 213-223.
","pubmedId":"","doi":"10.1002/biot.200900134"},{"reference":"de Lima JGS, Lanza DCF. 2021. 2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology. Viruses 13: 2160.
","pubmedId":"","doi":"10.3390/v13112160"},{"reference":"Fares H, Greenwald I. 2001. Genetic Analysis of Endocytosis in Caenorhabditis elegans: Coelomocyte Uptake Defective Mutants. Genetics 159: 133-145.
","pubmedId":"","doi":"10.1093/genetics/159.1.133"},{"reference":"Fitzgerald K, Greenwald I. 1995. Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo. Development 121: 4275-4282.
","pubmedId":"","doi":"10.1242/dev.121.12.4275"},{"reference":"Grant B, Greenwald I. 1997. Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans. Development 124: 637-644.
","pubmedId":"","doi":"10.1242/dev.124.3.637"},{"reference":"Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Embarek AM. 2018. Insights into Structural and Mechanistic Features of Viral IRES Elements. Frontiers in Microbiology 8: 10.3389/fmicb.2017.02629.
","pubmedId":"","doi":"10.3389/fmicb.2017.02629"},{"reference":"Nance J, Frøkjær-Jensen C. 2019. The Caenorhabditis elegans Transgenic Toolbox. Genetics 212: 959-990.
","pubmedId":"","doi":"10.1534/genetics.119.301506"},{"reference":"Ng JPZ, Mariati M, Bi J, Chang MW, Yang Y. 2025. A Targeted Integration-Based CHO Cell Platform for Simultaneous Antibody Display and Secretion. Antibodies 14: 38.
","pubmedId":"","doi":"10.3390/antib14020038"},{"reference":"Nonet ML. 2023. Rapid generation of Caenorhabditis elegans single-copy transgenes combining recombination-mediated cassette exchange and drug selection. GENETICS 224: 10.1093/genetics/iyad072.
","pubmedId":"","doi":"10.1093/genetics/iyad072"},{"reference":"Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP. 2012. Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide. PLoS ONE 7: e43349.
","pubmedId":"","doi":"10.1371/journal.pone.0043349"},{"reference":"Schwartz ML, Jorgensen EM. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics 202: 1277-1288.
","pubmedId":"","doi":"10.1534/genetics.115.184275"},{"reference":"Spieth J, Brooke G, Kuersten S, Lea K, Blumenthal T. 1993. Operons in C. elegans: Polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-532.
","pubmedId":"","doi":"10.1016/0092-8674(93)90139-h"},{"reference":"Sun H, Beets I, Schafer W, Hobert O. 2023. Comparing engineered nuclear-localized reporter cassettes. MicroPubl Biol 2023: 10.17912/micropub.biology.001014.
","pubmedId":"38021170","doi":""},{"reference":"Wang X, Marchisio MA. 2021. Synthetic polycistronic sequences in eukaryotes. Synthetic and Systems Biotechnology 6: 254-261.
","pubmedId":"","doi":"10.1016/j.synbio.2021.09.003"},{"reference":"Wu WS, Huang WC, Brown JS, Zhang D, Song X, Chen H, et al., Lee. 2018. pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans. Nucleic Acids Research 46: W43-W48.
","pubmedId":"","doi":"10.1093/nar/gky277"},{"reference":"Yan J, Wang H, Xu Q, Jain N, Toxavidis V, Tigges J, et al., Gao. 2010. Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression. Analytical Biochemistry 399: 144-146.
","pubmedId":"","doi":"10.1016/j.ab.2009.11.032"}],"title":"An F2A sequence permits correct localization of a secreted and a nuclear localized reporter in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aegorhinus vitulus","label":"Aegorhinus vitulus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aerococcus","label":"Aerococcus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alaimidae","label":"Alaimidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"allobates femoralis","label":"Allobates femoralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alnus glutinosa","label":"Alnus glutinosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa aestivalis","label":"Alosa aestivalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa pseudoharengus","label":"Alosa pseudoharengus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alternaria alternata","label":"Alternaria alternata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"amynthas agrestis","label":"Amynthas Agrestis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma caninum","label":"Ancylostoma caninum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma ceylanicum","label":"Ancylostoma ceylanicum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anemone multifida","label":"Anemone multifida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anguilla rostrata","label":"Anguilla rostrata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anisakis simplex","label":"Anisakis simplex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anomala albopilosa","label":"Anomala albopilosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arabidopsis","label":"Arabidopsis","imageSrc":"arabidopsis.png","imageAlt":"Arabidopsis graphic by Zoe Zorn CC BY 4.0","mod":"TAIR","modLink":"https://arabidopsis.org","linkVariable":""},{"value":"architeuthis dux","label":"Architeuthis dux","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arion vulgaris","label":"Arion vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"armeria","label":"Armeria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"artemia","label":"Artemia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arthrobacter sp.","label":"Arthrobacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia","label":"Ascaridia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia galli","label":"Ascaridia galli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"asparagopsis taxiformis","label":"Asparagopsis taxiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"astatotilapia burtoni","label":"Astatotilapia burtoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"avena sativa","label":"Avena sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aves","label":"Aves","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus","label":"Bacillus (firmicutes)","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus cereus","label":"Bacillus cereus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus mycoides","label":"Bacillus mycoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus subtilis","label":"Bacillus subtilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus thuringiensis","label":"Bacillus thuringiensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus toyonensis","label":"Bacillus toyonensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus wiedmannii","label":"Bacillus wiedmannii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteria","label":"Bacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteriophage","label":"Bacteriophage","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bactrocera","label":"Bactrocera sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"batrachospermum gelatinosum","label":"Batrachospermum gelatinosum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula lenta","label":"Betula lenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula nigra","label":"Betula nigra","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus dahlbohmii","label":"Bombus dahlbohmii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus terrestris","label":"Bombus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombyx mori","label":"Bombyx mori","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bos taurus","label":"Bos Taurus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brachygobius doriae","label":"Brachygobius doriae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica oleracea","label":"Brassica oleracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica rapa","label":"Brassica rapa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brugia malayi","label":"Brugia malayi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"burkholderia thailandensis","label":"Burkholderia thailandensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"buttiauxella","label":"Buttiauxella","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis brenneri","label":"Caenorhabditis brenneri","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis briggsae","label":"Caenorhabditis briggsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"c. elegans","label":"Caenorhabditis elegans","imageSrc":"c-elegans.jpg","imageAlt":"C. elegans graphic by Zoe Zorn CC BY 4.0","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"caenorhabditis inopinata","label":"Caenorhabditis inopinata","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis japonica","label":"Caenorhabditis japonica","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis nigoni","label":"Caenorhabditis nigoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis remanei","label":"Caenorhabditis remanei","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis tropicalis","label":"Caenorhabditis tropicalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus","label":"Calidifontibacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus erzuremensis","label":"Calidifontibacillus erzuremensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calliphora sp","label":"Calliphora sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caltha sagittata","label":"Caltha sagittata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cambarus latimanus","label":"Cambarus latimanus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"candida albicans","label":"Candida albicans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"canis familiaris","label":"Canis familiaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cannabis sativa","label":"Cannabis sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caretta caretta","label":"Caretta caretta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cassiopea xamachana","label":"Cassiopea xamachana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caulobacter vibrioides","label":"Caulobacter vibrioides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cephalopods","label":"Cephalopoda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cerastium arvense","label":"Cerastium arvense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceriodaphnia","label":"Ceriodaphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceroglossus suturalis","label":"Ceroglossus suturalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chaetoceros","label":"Chaetoceros","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chamaecrista fasciculata","label":"Chamaecrista fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chilicola chalcidiformis","label":"Chilicola chalcidiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chitinimonas","label":"Chitinimonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chlamydomonas reinhardtii","label":"Chlamydomonas reinhardtii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chromobacterium","label":"Chromobacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysemys picta","label":"Chrysemys picta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysoperla rufilabris","label":"Chrysoperla rufilabris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"citrus","label":"Citrus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"clavibacter sp.","label":"Clavibacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"colinus virginianus","label":"Colinus virginianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crassostrea virginica","label":"Crassostrea virginica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crithidia fasciculata","label":"Crithidia fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cutibacterium acnes","label":"Cutibacterium acnes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cyanobacteria","label":"Cyanobacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia","label":"Daphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia pulex","label":"Daphnia pulex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dermacoccus nishinomiyaensis","label":"Dermacoccus nishinomiyaensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera","label":"Diabrotica virgifera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera virgifera virus 1","label":"Diabrotica virgifera virgifera virus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"d. discoideum","label":"Dictyostelium discoideum","imageSrc":"dicty.png","imageAlt":"D. discoideum","mod":"dictyBase","modLink":"http://dictybase.org","linkVariable":""},{"value":"diptera","label":"Diptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dotocryptus bellicosus","label":"Dotocryptus bellicosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drechmeria coniospora","label":"Drechmeria coniospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drosophila","label":"Drosophila","imageSrc":"drosophila.png","imageAlt":"Drosophila graphic by Zoe Zorn CC BY 4.0","mod":"FlyBase","modLink":"https://flybase.org/doi/","linkVariable":"doi"},{"value":"dryopteris campyloptera","label":"Dryopteris campyloptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris expansa","label":"Dryopteris expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris intermedia","label":"Dryopteris intermedia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dugesia dorotocephala","label":"Dugesia dorotocephala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"elasmobranchii","label":"Elasmobranchii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"embryophyta","label":"Embryophyta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enoploteuthis chunii","label":"Enoploteuthis chunii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterobacter aerogenes","label":"Enterobacter aerogenes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterococcus raffinosus","label":"Enterococcus raffinosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"epichloë coenophiala","label":"Epichloë coenophiala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"equus caballus","label":"Equus caballus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erigeron sp","label":"Erigeron sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eristalis","label":"Eristalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eruca vesicaria","label":"Eruca vesicaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erwinia carotovora","label":"Erwinia carotovora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erythronium americanum","label":"Erythronium americanum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"escherichia coli","label":"Escherichia coli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eukaryota","label":"Eukaryotes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"felis catus","label":"Felis catus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella novicida","label":"Francisella novicida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella tularensis","label":"Francisella tularensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fraxinus americana","label":"Fraxinus americana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fucus distichus","label":"Fucus distichus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fungi","label":"Fungi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gasteropelecus sp.","label":"Gasteropelecus sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"geranium sp","label":"Geranium sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"girardia","label":"Girardia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glaucomys volans","label":"Glaucomys volans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glycine max","label":"Glycine max","imageSrc":"","imageAlt":"","mod":"Soybase","modLink":"https://soybase.org","linkVariable":""},{"value":"glyptemys insculpta","label":"Glyptemys insculpta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gossypium hirsutum","label":"Gossypium hirsutum","imageSrc":"","imageAlt":"","mod":"CottonGen","modLink":"https://www.cottongen.org/","linkVariable":""},{"value":"gromphadorhina portentosa","label":"Gromphadorhina portentosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gryllodes sigillatus","label":"Gryllodes sigillatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"haliotis rufescens","label":"Haliotis rufescens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hepacivirus hominis","label":"Hepatitis C Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"herpes simplex virus type 1","label":"Herpes simplex virus type 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human","label":"Human","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human coronavirus oc43","label":"Human coronavirus OC43","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydra vulgaris","label":"Hydra vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydropsyche sp","label":"Hydropsyche sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hymenoptera","label":"Hymenoptera","imageSrc":"","imageAlt":"","mod":"Hymenoptera Genome Database","modLink":"https://hymenoptera.elsiklab.missouri.edu/","linkVariable":""},{"value":"hypochaeris radicata","label":"Hypochaeris radicata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hypodynerus vespiformis","label":"Hypodynerus vespiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflaviridae","label":"Iflaviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflavuris","label":"Iflavirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ipomoea hederacea","label":"Ipomoea hederacea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera","label":"Ischnomera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera ruficollis","label":"Ischnomera ruficollis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"julidochromis marlieri","label":"Julidochromis marlieri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"juniperus virginiana","label":"Juniperus virginiana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"kluyveromyces marxianus","label":"Kluyveromyces marxianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"l. casei","label":"L. casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lacticaseibacillus casei","label":"Lacticaseibacillus casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lactobacillus","label":"Lactobacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"larentiinae sp","label":"Larentiinae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"laurus nobilis","label":"Laurus nobilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lepidoptera","label":"Lepidoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"leucanthemum vulgare","label":"Leucanthemum vulgare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ligilactobacillus","label":"Ligilactobacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ligilactobacillus salivarius","label":"Ligilactobacillus salivarius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"limosilactobacillus","label":"Limosilactobacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"linepithema humile","label":"Linepithema humile","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"liometopum occidentale","label":"Liometopum occidentale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lolium arundinaceum","label":"Lolium arundinaceum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lontra longicaudis","label":"Lontra longicaudis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbriculus variegatus","label":"Lumbriculus variegatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbricus terrestris","label":"Lumbricus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lupinus polyphyllus","label":"Lupinus polyphyllus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lycorma delicatula","label":"Lycorma delicatula","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lynx rufus","label":"Lynx rufus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"magnaporthe oryzae","label":"Magnaporthe oryzae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mammalia","label":"Mammalia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"manihot esculenta","label":"Manihot esculenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"medicago lupulina","label":"Medicago lupulina","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"meloidogyne","label":"Meloidogyne","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mimus polyglottos","label":"Mimus polyglottos","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bryophyta","label":"Mosses","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mouse","label":"Mouse","imageSrc":"","imageAlt":"","mod":"MGI","modLink":"https://informatics.jax.org","linkVariable":""},{"value":"m. minutoides","label":"Mus minutoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mycobacterium smegmatis","label":"Mycobacterium smegmatis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nakaseomyces glabratus","label":"Nakaseomyces glabratus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nauphoeta cinerea","label":"Nauphoeta cinerea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"neurospora","label":"Neurospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"n. benthamiana","label":"Nicotiana benthamiana","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/Nicotiana_benthamiana/genome","linkVariable":""},{"value":"nicotiana tabacum","label":"Nicotiana tabacum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae","label":"Noctuidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae sp","label":"Noctuidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nothobranchius furzeri","label":"Nothobranchius furzeri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"onchocerca volvulus","label":"Onchocerca volvulus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"orconectes virilis","label":"Orconectes virilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ormia ochracea","label":"Ormia ochracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"o. sativa","label":"Oryza sativa","imageSrc":"","imageAlt":"","mod":"Gramene","modLink":"https://www.gramene.org/","linkVariable":""},{"value":"other","label":"Other","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"oxalis enneaphylla","label":"Oxalis enneaphylla","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea","label":"Pantoea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea agglomerans","label":"Pantoea agglomerans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"papaver sp","label":"Papaver sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paramecium bursaria","label":"Paramecium bursaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"partitiviridae","label":"Partitiviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pelodiscus sinensis","label":"Pelodiscus sinensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"perezia recurvata","label":"Perezia recurvata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"petromyzon marinus","label":"Petromyzon marinus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis","label":"Photinus pyralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis associated partiti-like virus","label":"Photinus pyralis associated partiti-like virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis iflavirus 1","label":"Photinus pyralis iflavirus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"physcomitrium patens","label":"Physcomitrium patens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus strobus","label":"Pinus strobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus taeda","label":"Pinus taeda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"platycheirus","label":"Platycheirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"plectus sambesii","label":"Plectus sambesii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pogonomyrmex occidentalis","label":"Pogonomyrmex occidentalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"poncirus trifoliata","label":"Poncirus trifoliata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"populus deltoides","label":"Populus deltoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"potato virus y","label":"Potato virus Y","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"primula magellanica","label":"Primula magellanica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pristionchus pacificus","label":"Pristionchus pacificus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"prunus persica","label":"Prunus persica","imageSrc":"","imageAlt":"","mod":"Genome Database for Rosaceae","modLink":"https://www.rosaceae.org/","linkVariable":""},{"value":"psalmopoeus iriminia","label":"Psalmopoeus iriminia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudanabaena sp.","label":"Pseudanabaena sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas","label":"Pseudomonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas aeruginosa","label":"Pseudomonas aeruginosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas glycinae","label":"Pseudomonas glycinae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas putida","label":"Pseudomonas putida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas syringae","label":"Pseudomonas syringae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pterophyllum scalare","label":"Pterophyllum scalare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"python regius","label":"Python regius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"quercus macrocarpa","label":"Quercus macrocarpa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ralstonia solanacearum","label":"Ralstonia solanacearum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranitomeya imitator","label":"Ranitomeya imitator","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranunculus peduncularis","label":"Ranunculus peduncularis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"rat","label":"Rat","imageSrc":"","imageAlt":"","mod":"RGD","modLink":"https://rgd.mcw.edu","linkVariable":""},{"value":"rheinheimera","label":"Rheinheimera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ribes rubrum","label":"Ribes rubrum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"sars-cov-2","label":"SARS-CoV-2","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. cerevisiae","label":"Saccharomyces cerevisiae","imageSrc":"yeast.png","imageAlt":"Yeast graphic by Zoe Zorn CC BY 4.0","mod":"SGD","modLink":"https://yeastgenome.org","linkVariable":""},{"value":"saccharomyces paradoxus","label":"Saccharomyces paradoxus ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. uvarum","label":"Saccharomyces uvarum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schistosoma","label":"Schistosoma","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schizosaccharomyces japonicus","label":"Schizosaccharomyces japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. pombe","label":"Schizosaccharomyces pombe","imageSrc":"pombe.png","imageAlt":"Pombe graphic by Zoe Zorn © Caltech","mod":"PomBase","modLink":"https://www.pombase.org/reference/PMID:","linkVariable":"pmId"},{"value":"schmidtea mediterranea","label":"Schmidtea mediterranea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"senecio sp","label":"Senecio sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"simocephalus","label":"Simocephalus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"siraitia grosvenorii","label":"Siraitia grosvenorii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"solanum lycopersicum","label":"Solanum lycopersicum","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/1/view/","linkVariable":""},{"value":"sorghum","label":"Sorghum","imageSrc":"","imageAlt":"","mod":"SorghumBase","modLink":"https://www.sorghumbase.org","linkVariable":""},{"value":"spiroplasma eriocheiris","label":"Spiroplasma eriocheiris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus aureus","label":"Staphylococcus aureus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus epidermidis","label":"Staphylococcus epidermidis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"steinernema carpocapsae","label":"Steinernema carpocapsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"steinernema hermaphroditum","label":"Steinernema hermaphroditum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stenotrophomonas geniculata","label":"Stenotrophomonas geniculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stewartia floidana","label":"Stewartia floridana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus gordonii ","label":"Streptococcus gordonii ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus mutans","label":"Streptococcus mutans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":" streptococcus pneumoniae","label":"Streptococcus pneumoniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. purpuratus","label":"Strongylocentrotus purpuratus","imageSrc":"","imageAlt":"","mod":"Echinobase","modLink":"https://www.echinobase.org","linkVariable":""},{"value":"strongyloides ratti","label":"Strongyloides ratti","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"sulfolobus","label":"Sulfolobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"symphoricarpos albus","label":"Symphoricarpos albus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syncirsodes","label":"Syncirsodes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"synechococcus elongatus","label":"Synechococcus elongatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syrphidae","label":"Syrphidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tarantobelus jeffdanielsi","label":"Tarantobelus jeffdanielsi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"taraxacum officinale","label":"Taraxacum officinale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tatochila theodice","label":"Tatochila theodice","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetrahymena","label":"Tetrahymena","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetramorium immigrans","label":"Tetramorium immigrans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tomato brown rugose fruit virus","label":"ToBRFV","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trachemys scripta","label":"Trachemys scripta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tribolium castaneum","label":"Tribolium castaneum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichoptera","label":"Trichoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichuris muris","label":"Trichuris muris","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"trifolium repens","label":"Trifolium repens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trypoxylus dichotomus","label":"Trypoxylus dichotomus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tsuga canadensis","label":"Tsuga canadensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ulva expansa","label":"Ulva expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"universal","label":"Universal","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"vargula hilgendorfii","label":"Vargula hilgendorfii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"vespula vulgaris","label":"Vespula vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"virus","label":"Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"watasenia scintillans","label":"Watasenia scintillans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"wolbachia pipientis","label":"Wolbachia pipientis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"xenopus","label":"Xenopus","imageSrc":"xenopus.png","imageAlt":"Xenopus graphic by Zoe Zorn CC BY 4.0","mod":"XenBase","modLink":"https://xenbase.org","linkVariable":""},{"value":"xenorhabdus griffiniae","label":"Xenorhabdus griffiniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"yramea cytheris","label":"Yramea cytheris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zaprionus indianus","label":"Zaprionus indianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zea mays","label":"Zea mays","imageSrc":"","imageAlt":"","mod":"MaizeGDB","modLink":"https://www.maizegdb.org","linkVariable":""},{"value":"zebrafish","label":"Zebrafish","imageSrc":"zebrafish.png","imageAlt":"Zebrafish graphic by Zoe Zorn CC BY 4.0","mod":"ZFIN","modLink":"https://zfin.org","linkVariable":""}]}},"pageContext":{"id":"7727e5df-4092-4f9a-8638-f8ecf25c74e8","citedBy":[],"parsedCsv":{"csvHeader":[],"csvData":[]}}}, "staticQueryHashes": ["2114697108"]}