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Epstein-Barr virus-derived vector suitable for long-term expression in neurons

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Kiyosue, Kazuyuki, Miwa, Yoshihiro·2020·Heliyon
DOI 10.1016/j.heliyon.2020.e03504PMC PMC7068671
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Abstract

Exogenous gene expression is a fundamental and indispensable technique for testing gene function in neurons. Several ways to express exogenous genes in neurons are available, but each method has pros and cons. The lentivirus vector is useful for high efficiency gene transfer to neurons and stabilizes gene expression via genome integration, but this integration may destroy the host genome. The Epstein-Barr virus (EBV)-derived vector (EB vector) is an accessible and useful vector in human cell lines because the vector is not integrated into the host genome but stays in the nucleus as an episome. However, there has been no report on this process in rodent neurons. We examined the usefulness of the EB vector for testing gene function in neurons. We found that EB vector-derived exogenous proteins such as green fluorescent protein (GFP) and GFP-tagged actin were easily detectable even after three weeks of transfection. Second, a tetracycline-induced gene expression system in the EB vector was active after three weeks of transfection, indicating that plasmids were retained in neurons for up to three weeks. Third, we determined that only Family of repeat element of the plasmid vector is essential for its long-term presence in neurons. These results show that the modified EB vector is a useful tool for examining gene function in neurons.

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Figure 1 EBNA1 dependent long-term gene expression in neurons. A. Plasmid maps of vectors, EB vector and EBNA lacking EB vector (delta EBNA). B. A typical fluorescent images of GFP-actin transfected neurons cultured for 23 days (23DIV) with EB or delta EBNA vectors (Upper: GFP, Lower: Hoechst staining, scale bar 100μm). Transfections were done at 0 DIV and neurons were fix at 23DIV and stained. C. The number of GFP signal positive neurons and the intensity of the GFP fluorescence were significantly higher in case of EB vector than delta EBNA vector (GFP signal positive neurons; EB vector: 11.90 ± 1.39, n = 15, delta EBNA: 2.20 ± 0.66, n = 15, p < 0.005, GFP signal intensity; EB vector: 1943.7 ± 105.6, n = 689, delta EBNA: 306.1 ± 23.8, n = 696, p < 0.005). The number of fields and cells for quantification are indicated above each bar. D. mRNA comparison, Quantitative PCR shows high number of mRNA in EB vector transfected cultured neurons that delta EBNA vector transfected one.
Figure 1 EBNA1 dependent long-term gene expression in neurons. A. Plasmid maps of vectors, EB vector and EBNA lacking EB vector (delta EBNA). B. A typical fluorescent images of GFP-actin transfected neurons cultured for 23 days (23DIV) with EB or delta EBNA vectors (Upper: GFP, Lower: Hoechst staining, scale bar 100μm). Transfections were done at 0 DIV and neurons were fix at 23DIV and stained. C. The number of GFP signal positive neurons and the intensity of the GFP fluorescence were significantly higher in case of EB vector than delta EBNA vector (GFP signal positive neurons; EB vector: 11.90 ± 1.39, n = 15, delta EBNA: 2.20 ± 0.66, n = 15, p < 0.005, GFP signal intensity; EB vector: 1943.7 ± 105.6, n = 689, delta EBNA: 306.1 ± 23.8, n = 696, p < 0.005). The number of fields and cells for quantification are indicated above each bar. D. mRNA comparison, Quantitative PCR shows high number of mRNA in EB vector transfected cultured neurons that delta EBNA vector transfected one.CC BY-NC-ND · PubMed Central
Figure 2 Long term plasmid retention in neurons. A. Cartoon of DOX induction plasmids system. EBNA1 and TetOn3G were expressed by pEB6_CAG_Teton3G vector (upper) and the complex of expressed 3G and DOX binds to pOrip_3GBi_GFP vector (lower) to activate expression of GFP and Gene of Interest (GOI) expression. B. Induction of GFP expression by DOX addition. There were no GFP positive neurons before DOX induction (upper 4 panels), but adding DOX for 24h induced GFP expression in a number of neurons (lower 4 panels).
Figure 2 Long term plasmid retention in neurons. A. Cartoon of DOX induction plasmids system. EBNA1 and TetOn3G were expressed by pEB6_CAG_Teton3G vector (upper) and the complex of expressed 3G and DOX binds to pOrip_3GBi_GFP vector (lower) to activate expression of GFP and Gene of Interest (GOI) expression. B. Induction of GFP expression by DOX addition. There were no GFP positive neurons before DOX induction (upper 4 panels), but adding DOX for 24h induced GFP expression in a number of neurons (lower 4 panels).CC BY-NC-ND · PubMed Central
Figure 3 Identification of critical component of OriP sequence for retention in neurons. A. Cartoon of the pOriPdelta_3GBi_GFP plasmid vector and major components of OriP fragment. Deletion variants are indicated (lower). B. GFP expression from full length vector and deletion clones were compared. Inductions of GFP expression were done 3 weeks after transfection. Deleting FR component from OriP fragment are dramatically reduced the number of GFP positive neurons (delta DS: 81.71 ± 11.83% of full length, n = 5 trial, delta DS-L: 57.71 ± 7.35%, n = 4 trial, delta FR: 12.72 ± 7.74%, n = 5 trial. Full vs. delta DS: p = 0.335, Full vs. delta DS-L, p = 0.037, Full vs. delta FR, p = 0.00273, steel-Dwass test).
Figure 3 Identification of critical component of OriP sequence for retention in neurons. A. Cartoon of the pOriPdelta_3GBi_GFP plasmid vector and major components of OriP fragment. Deletion variants are indicated (lower). B. GFP expression from full length vector and deletion clones were compared. Inductions of GFP expression were done 3 weeks after transfection. Deleting FR component from OriP fragment are dramatically reduced the number of GFP positive neurons (delta DS: 81.71 ± 11.83% of full length, n = 5 trial, delta DS-L: 57.71 ± 7.35%, n = 4 trial, delta FR: 12.72 ± 7.74%, n = 5 trial. Full vs. delta DS: p = 0.335, Full vs. delta DS-L, p = 0.037, Full vs. delta FR, p = 0.00273, steel-Dwass test).CC BY-NC-ND · PubMed Central
Figure 4 Exogenous gene products, GluR2Q, alters synaptic property. A. Typical spontaneous synaptic currents recorded from transfected neurons with holding potentials at -70 and +40mV presented in lower and upper waveform, respectively. Synaptic currents at +40mV from GluA2 expressing neuron are greatly reduced compared to the control one. This inward rectification of synaptic currents means GluA2Q subunit is incorporated into synaptic glutamate receptors. B. Summary data of rectification index are plotted (control: 0.731 ± 0.067, n = 13 cells, GluA2Q: 0.456 ± 0.05, n = 10 cells).
Figure 4 Exogenous gene products, GluR2Q, alters synaptic property. A. Typical spontaneous synaptic currents recorded from transfected neurons with holding potentials at -70 and +40mV presented in lower and upper waveform, respectively. Synaptic currents at +40mV from GluA2 expressing neuron are greatly reduced compared to the control one. This inward rectification of synaptic currents means GluA2Q subunit is incorporated into synaptic glutamate receptors. B. Summary data of rectification index are plotted (control: 0.731 ± 0.067, n = 13 cells, GluA2Q: 0.456 ± 0.05, n = 10 cells).CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 21

  1. Lentz, 2012, Viral vectors for gene delivery to the central nervous system, Neurobiol. Dis., 48, 179, 10.1016/j.nbd.2011.09.014 · DOI
  2. Karra, 2010, Transfection techniques for neuronal cells, J. Neurosci., 30, 6171, 10.1523/JNEUROSCI.0183-10.2010 · DOI · Paper page
  3. Davidson, 2003, Viral vectors for gene delivery to the nervous system, Nat. Rev. Neurosci., 4, 353, 10.1038/nrn1104 · DOI
  4. Yates, 1984, A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells, Proc. Natl. Acad. Sci. U.S.A., 81, 3806, 10.1073/pnas.81.12.3806 · DOI
  5. Yates, 1985, Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells, Nature, 313, 812, 10.1038/313812a0 · DOI
  6. Lupton, 1985, Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells, Mol. Cell Biol., 5, 2533
  7. Tanaka, 1999, Construction of Epstein-Barr virus-based expression vector containing mini-oriP, Biochem. Biophys. Res. Commun., 264, 938, 10.1006/bbrc.1999.1617 · DOI
  8. Lufino, 2008, Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression, Mol. Ther., 16, 1525, 10.1038/mt.2008.156 · DOI
  9. Rawlins, 1985, Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region, Cell, 42, 859, 10.1016/0092-8674(85)90282-X · DOI
  10. Nakayama, 2005, Diminished neuronal activity increases neuron-neuron connectivity underlying silent synapse formation and the rapid conversion of silent to functional synapses, J. Neurosci., 25, 4040, 10.1523/JNEUROSCI.4115-04.2005 · DOI
  11. Edelstein, 2014, Advanced methods of microscope control using μManager software, J. Biol. Methods, 1, 10
  12. Hayes, 2018, Computer-aided neurophysiology and imaging with open-source PhysImage, J. Neurophysiol., 120, 23, 10.1152/jn.00048.2017 · DOI
  13. Fan, 2012, Transient, inducible, placenta-specific gene expression in mice, Endocrinology, 153, 5637, 10.1210/en.2012-1556 · DOI
  14. Akamatsu, 2016, The AMPA receptor antagonist perampanel robustly rescues amyotrophic lateral sclerosis (ALS) pathology in sporadic ALS model mice, Sci. Rep., 6, 28649, 10.1038/srep28649 · DOI
  15. Tateno, 2004, Calcium-permeable AMPA receptors promote misfolding of mutant SOD1 protein and development of amyotrophic lateral sclerosis in a transgenic mouse model, Hum. Mol. Genet., 13, 2183, 10.1093/hmg/ddh246 · DOI
  16. Bowie, 1995, Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block, Neuron, 15, 453, 10.1016/0896-6273(95)90049-7 · DOI
  17. Shi, 2001, Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons, Cell, 105, 331, 10.1016/S0092-8674(01)00321-X · DOI · Paper page
  18. Wysokenski, 1989, Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus, J. Virol., 63, 2657, 10.1128/jvi.63.6.2657-2666.1989 · DOI
  19. Reisinger, 2006, Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization, Int. J. Canc., 118, 1603, 10.1002/ijc.21498 · DOI
  20. Howard, 2008, Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro, Virology, 372, 24, 10.1016/j.virol.2007.10.007 · DOI · Paper page
  21. Lo, 2009, Development of a hybrid baculoviral vector for sustained transgene expression, Mol. Ther., 17, 658, 10.1038/mt.2009.13 · DOI

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Metadata

Authors
Kiyosue, K., & Miwa, Y.
Affiliations
  • National Institute of Advanced Industrial Science and Technology (JP)
  • University of Tsukuba (JP)
Corresponding author
Kiyosue, Kazuyuki
ORCID
Venue
Heliyon
Publisher
Elsevier BV
Published
2020-03
Volume
6
Issue
3
Pages
e03504
ISSN
2405-8440 (ISSN-L)
DOI
10.1016/j.heliyon.2020.e03504
PMC
PMC7068671
OpenAlex
W3011573275
Cited by
8
References
0
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2023: 1 · 2022: 1 · 2021: 2
FWCI
0.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Heliyon (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Japan Society for the Promotion of Science (15H04281, 16H01630, 16K14589, JP24500400)
  • National Institute of Advanced Industrial Science and Technology
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords
episomes, tetracycline-inducible expression, plasmid retention, GFP expression, transfection

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Kiyosue, K., & Miwa, Y. (2020). Epstein-Barr virus-derived vector suitable for long-term expression in neurons. Heliyon. https://doi.org/10.1016/j.heliyon.2020.e03504