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<document id="04B6695EB3BE4F22BD1FE80A28FA900F" ID-DOI="10.1093/zoolinnean/zlad196" ID-ISSN="0024-4082" ID-Zenodo-Dep="14423105" IM.bibliography_approvedBy="carolina" IM.illustrations_approvedBy="carolina" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="carolina" IM.tables_requiresApprovalFor="GgImagineBatch" IM.taxonomicNames_approvedBy="carolina" IM.treatments_approvedBy="carolina" checkinTime="1734029342775" checkinUser="plazi" docAuthor="Fu, Jinyu, Gao, Yunyi, Gao, Feng, Sheng, Yalan, Al-Farraj, Saleh A., Chen, Zigui &amp; Wang, Chundi" docDate="2024" docId="D06287D4FFB2FF9EFC1E05A3FE87FEE3" docLanguage="en" docName="zlad196.pdf" docOrigin="Zoological Journal of the Linnean Society 202 (3)" docSource="https://doi.org/10.1093/zoolinnean/zlad196" docStyle="DocumentStyle:4F230B9370E98E256D973D6DFB57F36C.9:ZoolJLinnSoc.2023-.journal_article" docStyleId="4F230B9370E98E256D973D6DFB57F36C" docStyleName="ZoolJLinnSoc.2023-.journal_article" docStyleVersion="9" docTitle="Paramecium tetraurelia" docType="treatment" docVersion="1" lastPageNumber="9" masterDocId="2C5BFFACFFBAFF97FFDF0149FC7DFFE5" masterDocTitle="An overview of genetic manipulation tools in the studies of ciliates * with emphasis on gene knockout * knockdown * and overexpression" masterLastPageNumber="13" masterPageNumber="1" pageNumber="9" updateTime="1734439180940" updateUser="carolina">
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<mods:namePart id="A1D1274DBE2C030FBC7C018535EEEE13">Gao, Yunyi</mods:namePart>
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<paragraph id="587436C2FFB2FF9FFC1E05A3F947FAE0" blockId="8.[825,1475,1258,2010]" box="[961,1338,1258,1285]" pageId="8" pageNumber="9">
<emphasis id="6ABFEAD0FFB2FF9FFC1E05A3F947FAE0" bold="true" box="[961,1338,1258,1285]" pageId="8" pageNumber="9">
Gene overexpression in
<taxonomicName id="9FCB4D41FFB2FF9FFB6105A2F947FAE0" authority="Sonneborn" box="[1214,1338,1258,1285]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="tetraurelia">
<emphasis id="6ABFEAD0FFB2FF9FFB6105A2F947FAE0" bold="true" box="[1214,1338,1258,1285]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
</taxonomicName>
</emphasis>
</paragraph>
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<subSubSection id="10D16549FFB2FF9EFCE60458FE87FEE3" lastPageId="9" lastPageNumber="10" pageId="8" pageNumber="9" type="description">
<paragraph id="587436C2FFB2FF9FFCE60458F873F964" blockId="8.[825,1475,1258,2010]" pageId="8" pageNumber="9">
Gene overexpression can be achieved by introducing more copies of the target gene into the MAC in
<taxonomicName id="9FCB4D41FFB2FF9FFB010478F925FAAD" box="[1246,1368,1328,1352]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="tetraurelia">
<emphasis id="6ABFEAD0FFB2FF9FFB010478F925FAAD" box="[1246,1368,1328,1352]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
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(Table 1). A small portion of exogenous DNA introduced into the MAC of
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<emphasis id="6ABFEAD0FFB2FF9FFC8B0439FFB2FA62" box="[852,975,1391,1415]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
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can be integrated into the chromosomes, similar to
<taxonomicName id="9FCB4D41FFB2FF9FFC8C04C7FFAEFA43" box="[851,979,1422,1446]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFC8C04C7FFAEFA43" box="[851,979,1422,1446]" italics="true" pageId="8" pageNumber="9">Tetrahymena</emphasis>
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, while most is added with
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<emphasis id="6ABFEAD0FFB2FF9FFB3E04C6F926FA43" box="[1249,1371,1423,1446]" italics="true" pageId="8" pageNumber="9">Paramecium</emphasis>
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telomerelike sequences at both termini and replicates autonomously (
<bibRefCitation id="3C5A4B33FFB2FF9FFC9C0484FF88FA00" author="Gilley" box="[835,1013,1485,1509]" etAl="et al." firstAuthor="Gilley" pageId="8" pageNumber="9" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
Gilley
<emphasis id="6ABFEAD0FFB2FF9FFC560487FFC6FA00" box="[905,955,1485,1509]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
1988
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, Bourgain and Katinka 1991). Not only the linear DNA but also the circular DNA can be maintained by cleavage at non-random sites to convert it from supercoiled to linear (
<bibRefCitation id="3C5A4B33FFB2FF9FFC5C0762F83AF9A6" author="Godiska" box="[899,1095,1579,1603]" etAl="et al." firstAuthor="Godiska" pageId="8" pageNumber="9" pagination="7590 - 4" refId="ref9827" refString="Godiska R * Aufderheide KJ * Gilley D et al. ºransformation of Paramecium by microinjection of a cloned serotype gene. Proceedings of the National Academy of Sciences of the United States of America 1987; 84: 7590 - 4. hups: // doi. org / 10.1073 / pnas. 84.21.7590" type="journal article" year="1987">
Godiska
<emphasis id="6ABFEAD0FFB2FF9FFC000765F873F9A6" box="[991,1038,1579,1603]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
1987
</bibRefCitation>
,
<bibRefCitation id="3C5A4B33FFB2FF9FFB8D0762F882F9A6" author="Gilley" box="[1106,1279,1579,1603]" etAl="et al." firstAuthor="Gilley" pageId="8" pageNumber="9" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
Gilley
<emphasis id="6ABFEAD0FFB2FF9FFB490765F8BBF9A6" box="[1174,1222,1579,1603]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
1988
</bibRefCitation>
). To guarantee the efficiency of retention of exogenous DNA, linear DNA is used in routine experiments.
</paragraph>
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The flanking sequences and target gene are amplified and inserted into the vector (
<figureCitation id="C0F02A47FFB2FF9FFBF007E1F804F925" box="[1071,1145,1704,1728]" captionStart="Figure 5" captionStartId="8.[129,194,1018,1042]" captionTargetBox="[138,1466,153,980]" captionTargetId="figure-518@8.[128,1475,144,991]" captionTargetPageId="8" captionText="Figure 5. Gene overexpression strategies in ciliates.A* construct of gene overexpression and the homologous recombination in Tetrahymena (a) and positive transformed cell screening (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing to the plasmid indicate the restriction enzyme digestion sites. B* gene overexpression by injection of more copies in Paramecium. ºelomeres are indicated with red squares at the end of chromosomes. C* gene overexpression by introduction of artificial chromosomes in Oxytricha. Abbreviations:GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; MT1 and MT3* cadmiuminducible metallothionein genes; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin." figureDoi="http://doi.org/10.5281/zenodo.14423119" httpUri="https://zenodo.org/record/14423119/files/figure.png" pageId="8" pageNumber="9">Fig. 5B</figureCitation>
). To meet the requirements of some experiments, a tag sequence can be added immediately after the start codon or before the stop codon of the target gene. The plasmid is linearized by restriction enzyme digestion at the backbone region, then introduced into the MAC of vegetative cells by microinjection. Positive injected cells can be checked using PCR or dot-blot by specifically detecting the DNA that is not present in the genome of
<taxonomicName id="9FCB4D41FFB2FF9FFB8606CDF8AEF87E" box="[1113,1235,1924,1947]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFB8606CDF8AEF87E" box="[1113,1235,1924,1947]" italics="true" pageId="8" pageNumber="9">Paramecium</emphasis>
</taxonomicName>
. Owing to the high concentration of DNA microinjected, the target gene in the transformed cells is prominently overexpressed compared with the wild-type cells. Moreover, the injected linear constructs can be maintained for a long time in vegetative cells without decreasing rapidly during cell division (
<bibRefCitation id="3C5A4B33FFB3FF9EFE490186FE3EFF02" author="Gilley" box="[406,579,207,231]" etAl="et al." firstAuthor="Gilley" pageId="9" pageNumber="10" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
Gilley
<emphasis id="6ABFEAD0FFB3FF9EFE050186FE77FF02" box="[474,522,207,231]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1988
</bibRefCitation>
). However, DNA is lost with the degradation of the old MAC during conjugation.
</paragraph>
</subSubSection>
</treatment>
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<mods:affiliation id="4160AFA0C0BF507E581E504A5BBDB5DC">Guangdong Key Laboratory of Subtropical Biodiversity and Biomonitoring * School of Life Sciences * South China Normal University * Guangzhou 510631 * China</mods:affiliation>
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<mods:affiliation id="08574885A40BAE6900AEB03750550755">Zoology Department * College of Science * King Saud University * Riyadh 11451 * Saudi Arabia</mods:affiliation>
</mods:name>
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<mods:namePart id="B16635F925B81D5F6B662B59ED2BC649">Chen, Zigui</mods:namePart>
<mods:affiliation id="4CB718E5726DAFBF03DE81D4181599C3">Institute of Evolution &amp; Marine Biodiversity * Ocean University of China * Qingdao 266003 * China &amp; Key Laboratory of Evolution &amp; Marine Biodiversity (Ministry of Education) * Ocean University of China * Qingdao 266003 * China</mods:affiliation>
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<mods:namePart id="CB3164FF0CC1B1BC3E55319D794478D9">Wang, Chundi</mods:namePart>
<mods:affiliation id="A1E053A6896E609AF6F9F183215C4C64">Laboratory of Marine Protozoan Biodiversity &amp; Evolution * Marine College * Shandong University * Weihai 264209 * China</mods:affiliation>
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<mods:title id="E0DFDF9169338634A7E1DAB4B11D8B77">Zoological Journal of the Linnean Society</mods:title>
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<mods:part id="814B8E6957D2DFC1647CD54FAD41DC08">
<mods:date id="87C78C470CCFCFB307C9EA4F0167F1EE">2024</mods:date>
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<treatment id="D06287D4FFB3FF9EFEDF006CFD68F98D" LSID="urn:lsid:plazi:treatment:D06287D4FFB3FF9EFEDF006CFD68F98D" httpUri="http://treatment.plazi.org/id/D06287D4FFB3FF9EFEDF006CFD68F98D" lastPageNumber="10" pageId="9" pageNumber="10">
<subSubSection id="10D16549FFB3FF9EFEDF006CFE16FEA5" box="[256,619,293,320]" pageId="9" pageNumber="10" type="nomenclature">
<paragraph id="587436C2FFB3FF9EFEDF006CFE16FEA5" blockId="9.[113,764,293,1640]" box="[256,619,293,320]" pageId="9" pageNumber="10">
<emphasis id="6ABFEAD0FFB3FF9EFEDF006CFE16FEA5" bold="true" box="[256,619,293,320]" pageId="9" pageNumber="10">
Gene overexpression in
<taxonomicName id="9FCB4D41FFB3FF9EFE22006CFE16FEA5" ID-CoL="75GSZ" authority="Greslin et al., 1989" box="[509,619,293,320]" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="trifallax">
<emphasis id="6ABFEAD0FFB3FF9EFE22006CFE16FEA5" bold="true" box="[509,619,293,320]" italics="true" pageId="9" pageNumber="10">O. trifallax</emphasis>
</taxonomicName>
</emphasis>
</paragraph>
</subSubSection>
<subSubSection id="10D16549FFB3FF9EFFAE0005FD68F98D" pageId="9" pageNumber="10" type="description">
<paragraph id="587436C2FFB3FF9EFFAE0005FE00FCDF" blockId="9.[113,764,293,1640]" pageId="9" pageNumber="10">
Benefitting from the gene-sized nano-chromosomes in the macronuclear genome, gene overexpression in
<taxonomicName id="9FCB4D41FFB3FF9EFD800025FEB3FE66" box="[607,718,363,387]" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="trifallax">
<emphasis id="6ABFEAD0FFB3FF9EFD800025FEB3FE66" box="[607,718,363,387]" italics="true" pageId="9" pageNumber="10">O. trifallax</emphasis>
</taxonomicName>
can be achieved by introduction of synthetic artificial chromosomes (Table 1; Swart
<emphasis id="6ABFEAD0FFB3FF9EFED000E2FD40FE27" box="[271,317,426,450]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2013).
<bibRefCitation id="3C5A4B33FFB3FF9EFE5700E3FE3DFE27" author="Clay" box="[392,576,426,450]" etAl="et al." firstAuthor="Clay" pageId="9" pageNumber="10" pagination="3119 - 27" refId="ref8879" refString="Clay DM * Kim H * Landweber LF. ºransformation with artificial chromosomes in Oxytricha trifallax and their applications. G 3 (Bethesda Md.) 2019 a; 9: 3119 - 27. hups: // doi. org / 10.1534 / g 3.119.400298" type="journal article" year="2019" yearSuffix="a">
Clay
<emphasis id="6ABFEAD0FFB3FF9EFE6400E2FD95FE27" box="[443,488,426,450]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
(2019a)
</bibRefCitation>
successfully transferred an artificial chromosome, comprising complete flanking sequences, a target gene, and 20 bp of telomere sequences at each end, into the MAC of vegetative cells by microinjection to obtain functional proteins. The injected chromosomes were stably maintained in cells (over 40 generations) and were largely free of end erosion, even after encystment/excystment (
<bibRefCitation id="3C5A4B33FFB3FF9EFD6B032FFCADFD78" author="Clay" etAl="et al." firstAuthor="Clay" pageId="9" pageNumber="10" pagination="3119 - 27" refId="ref8879" refString="Clay DM * Kim H * Landweber LF. ºransformation with artificial chromosomes in Oxytricha trifallax and their applications. G 3 (Bethesda Md.) 2019 a; 9: 3119 - 27. hups: // doi. org / 10.1534 / g 3.119.400298" type="journal article" year="2019" yearSuffix="a">
Clay
<emphasis id="6ABFEAD0FFB3FF9EFD36032EFCF7FD78" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2019a
</bibRefCitation>
). It is noteworthy that the introduction of synthetic DNA homologous to an endogenous nano-chromosome into
<taxonomicName id="9FCB4D41FFB3FF9EFD3C03ECFCBDFD39" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="trifallax">
<emphasis id="6ABFEAD0FFB3FF9EFD3C03ECFCBDFD39" italics="true" pageId="9" pageNumber="10">O. trifallax</emphasis>
</taxonomicName>
during gene rearrangement resulted in decreased rather than increased expression of the corresponding genes, suggesting that the import of artificial chromosomes for overexpression should be at the vegetative stage (
<bibRefCitation id="3C5A4B33FFB3FF9EFE19026BFE11FCDF" author="Clay" box="[454,620,802,826]" etAl="et al." firstAuthor="Clay" pageId="9" pageNumber="10" pagination="3105 - 18" refId="ref8926" refString="Clay DM * Yerlici Vº * Villano DJ et al. Programmed chromosome deletion in the ciliate Oxytricha trifallax. G 3 (Bethesda Md.) 2019 b; 9: 3105 - 18. hups: // doi. org / 10.1534 / g 3.118.200930" type="journal article" year="2019" yearSuffix="b">
Clay
<emphasis id="6ABFEAD0FFB3FF9EFE25026AFE54FCDF" box="[506,553,802,826]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2019b
</bibRefCitation>
).
</paragraph>
<paragraph id="587436C2FFB3FF9EFF520208FE1EFB34" blockId="9.[113,764,293,1640]" pageId="9" pageNumber="10">
Artificial chromosome transformation has also been reported in
<taxonomicName id="9FCB4D41FFB3FF9EFF540228FCA0FC9C" box="[139,221,865,889]" class="Hypotrichea" family="Euplotidae" genus="Euplotes" kingdom="Chromista" order="Euplotida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFF540228FCA0FC9C" box="[139,221,865,889]" italics="true" pageId="9" pageNumber="10">Euplotes</emphasis>
</taxonomicName>
and
<taxonomicName id="9FCB4D41FFB3FF9EFED20228FD00FC9C" box="[269,381,865,889]" class="Hypotrichea" family="Oxytrichidae" genus="Stylonychia" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFED20228FD00FC9C" box="[269,381,865,889]" italics="true" pageId="9" pageNumber="10">Stylonychia</emphasis>
</taxonomicName>
(Table 1), where most chromosomes have the same gene size as
<taxonomicName id="9FCB4D41FFB3FF9EFE5302C9FEA5FC7D" authority="(Swanton et al. 1980)" baseAuthorityName="Swanton" baseAuthorityYear="1980" box="[396,728,896,920]" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFE5302C9FD92FC7D" box="[396,495,896,920]" italics="true" pageId="9" pageNumber="10">Oxytricha</emphasis>
(Swanton
<emphasis id="6ABFEAD0FFB3FF9EFDBE02C8FEEFFC7D" box="[609,658,896,920]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1980)
</taxonomicName>
. In
<taxonomicName id="9FCB4D41FFB3FF9EFFAE02D6FD0BFC52" authority="Dujardin" authorityName="Dujardin" box="[113,374,927,951]" class="Hypotrichea" family="Euplotidae" genus="Euplotes" kingdom="Chromista" order="Euplotida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="crassus">
<emphasis id="6ABFEAD0FFB3FF9EFFAE02D6FD6FFC52" box="[113,274,927,951]" italics="true" pageId="9" pageNumber="10">Euplotes crassus</emphasis>
Dujardin
</taxonomicName>
, 1841, artificial chromosomes were transferred into the newly developed MAC (72–75 h after forming pairs) by microinjection (
<bibRefCitation id="3C5A4B33FFB3FF9EFE110297FEEBFC13" author="Erbeznik" box="[462,662,990,1014]" etAl="et al." firstAuthor="Erbeznik" pageId="9" pageNumber="10" pagination="206 - 16" refId="ref9179" refString="Erbeznik M * Yao MC * Jahn CL. Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle. Journal of Eukaryotic Microbiology 1999; 46: 206 - 16. hups: // doi. org / 10.1111 / j. 1550 - 7408.1999. tb 04605. x" type="journal article" year="1999">
Erbeznik
<emphasis id="6ABFEAD0FFB3FF9EFDF00296FE20FC13" box="[559,605,990,1014]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999
</bibRefCitation>
) and into vegetative cells with the aid of non-liposomal lipid Effectene (Qiagen, Hilden,
<collectingCountry id="20DC7652FFB3FF9EFEFA0554FDF5FBD0" box="[293,392,1053,1077]" name="Germany" pageId="9" pageNumber="10">Germany</collectingCountry>
) (
<bibRefCitation id="3C5A4B33FFB3FF9EFE400554FE25FBD0" author="Bender" box="[415,600,1053,1077]" etAl="et al." firstAuthor="Bender" pageId="9" pageNumber="10" pagination="3168 - 72" refId="ref8245" refString="Bender J * Kampfer M * Klein A. Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus. Nucleic Acids Research 1999; 27: 3168 - 72. hups: // doi. org / 10.1093 / nar / 27.15.3168" type="journal article" year="1999">
Bender
<emphasis id="6ABFEAD0FFB3FF9EFE2F0554FE62FBD0" box="[496,543,1053,1077]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999
</bibRefCitation>
); in both cases, the introduced DNA could be stably maintained and expressed (
<bibRefCitation id="3C5A4B33FFB3FF9EFFA30512FD49FB96" author="Bender" box="[124,308,1115,1139]" etAl="et al." firstAuthor="Bender" pageId="9" pageNumber="10" pagination="3168 - 72" refId="ref8245" refString="Bender J * Kampfer M * Klein A. Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus. Nucleic Acids Research 1999; 27: 3168 - 72. hups: // doi. org / 10.1093 / nar / 27.15.3168" type="journal article" year="1999">
Bender
<emphasis id="6ABFEAD0FFB3FF9EFF130515FC86FB96" box="[204,251,1115,1139]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999
</bibRefCitation>
,
<bibRefCitation id="3C5A4B33FFB3FF9EFEE00512FE75FB96" author="Erbeznik" box="[319,520,1115,1139]" etAl="et al." firstAuthor="Erbeznik" pageId="9" pageNumber="10" pagination="206 - 16" refId="ref9179" refString="Erbeznik M * Yao MC * Jahn CL. Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle. Journal of Eukaryotic Microbiology 1999; 46: 206 - 16. hups: // doi. org / 10.1111 / j. 1550 - 7408.1999. tb 04605. x" type="journal article" year="1999">
Erbeznik
<emphasis id="6ABFEAD0FFB3FF9EFE7E0515FDADFB96" box="[417,464,1115,1139]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999
</bibRefCitation>
). In
<taxonomicName id="9FCB4D41FFB3FF9EFDE50512FE88FB96" box="[570,757,1115,1139]" class="Hypotrichea" family="Oxytrichidae" genus="Stylonychia" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="lemnae">
<emphasis id="6ABFEAD0FFB3FF9EFDE50512FE88FB96" box="[570,757,1115,1139]" italics="true" pageId="9" pageNumber="10">Stylonychia lemnae</emphasis>
</taxonomicName>
, injected artificial chromosomes could be stably maintained for ≥80 generations, with expression similar to that of the endogenous genes (Skovorodkin
<emphasis id="6ABFEAD0FFB3FF9EFE7305F3FDA6FB34" box="[428,475,1209,1233]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999, 2001).
</paragraph>
<paragraph id="587436C2FFB3FF9EFF520590FE87FA68" blockId="9.[113,764,293,1640]" pageId="9" pageNumber="10">
In
<taxonomicName id="9FCB4D41FFB3FF9EFF760590FD71FB14" box="[169,268,1241,1265]" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFF760590FD71FB14" box="[169,268,1241,1265]" italics="true" pageId="9" pageNumber="10">Oxytricha</emphasis>
</taxonomicName>
,
<taxonomicName id="9FCB4D41FFB3FF9EFEC70590FD17FB14" box="[280,362,1241,1265]" class="Hypotrichea" family="Euplotidae" genus="Euplotes" kingdom="Chromista" order="Euplotida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFEC70590FD17FB14" box="[280,362,1241,1265]" italics="true" pageId="9" pageNumber="10">Euplotes</emphasis>
</taxonomicName>
, and
<taxonomicName id="9FCB4D41FFB3FF9EFE7B0590FE69FB14" box="[420,532,1241,1265]" class="Hypotrichea" family="Oxytrichidae" genus="Stylonychia" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFE7B0590FE69FB14" box="[420,532,1241,1265]" italics="true" pageId="9" pageNumber="10">Stylonychia</emphasis>
</taxonomicName>
, the artificial chromosome can replicate as efficiently as endogenous ones (
<bibRefCitation id="3C5A4B33FFB3FF9EFD4605B1FCB8FACA" author="Bender" etAl="et al." firstAuthor="Bender" pageId="9" pageNumber="10" pagination="3168 - 72" refId="ref8245" refString="Bender J * Kampfer M * Klein A. Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus. Nucleic Acids Research 1999; 27: 3168 - 72. hups: // doi. org / 10.1093 / nar / 27.15.3168" type="journal article" year="1999">
Bender
<emphasis id="6ABFEAD0FFB3FF9EFD3605B0FCF7FACA" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
1999
</bibRefCitation>
, Skovorodkin
<emphasis id="6ABFEAD0FFB3FF9EFE810451FDF2FACA" box="[350,399,1303,1327]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2001,
<bibRefCitation id="3C5A4B33FFB3FF9EFE08045EFEF8FACA" author="Clay" box="[471,645,1303,1327]" etAl="et al." firstAuthor="Clay" pageId="9" pageNumber="10" pagination="3119 - 27" refId="ref8879" refString="Clay DM * Kim H * Landweber LF. ºransformation with artificial chromosomes in Oxytricha trifallax and their applications. G 3 (Bethesda Md.) 2019 a; 9: 3119 - 27. hups: // doi. org / 10.1534 / g 3.119.400298" type="journal article" year="2019" yearSuffix="a">
Clay
<emphasis id="6ABFEAD0FFB3FF9EFDD10451FE42FACA" box="[526,575,1303,1327]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2019a
</bibRefCitation>
); however, unlike
<taxonomicName id="9FCB4D41FFB3FF9EFF65047EFD49FAAB" box="[186,308,1335,1358]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB3FF9EFF65047EFD49FAAB" box="[186,308,1335,1358]" italics="true" pageId="9" pageNumber="10">Paramecium</emphasis>
</taxonomicName>
, telomere sequences are essential to them, and the construct without telomeres is degraded soon after being injected into
<taxonomicName id="9FCB4D41FFB3FF9EFEEC043CFE8BFA68" authority="(Skovorodkin et al. 2001)" baseAuthorityName="Skovorodkin" baseAuthorityYear="2001" box="[307,758,1397,1421]" class="Hypotrichea" family="Oxytrichidae" genus="Stylonychia" kingdom="Chromista" order="Oxytrichida" pageId="9" pageNumber="10" phylum="Ciliophora" rank="species" species="lemnae">
<emphasis id="6ABFEAD0FFB3FF9EFEEC043CFD91FA68" box="[307,492,1397,1421]" italics="true" pageId="9" pageNumber="10">Stylonychia lemnae</emphasis>
(Skovorodkin
<emphasis id="6ABFEAD0FFB3FF9EFD5B043FFECFFA68" box="[644,690,1397,1421]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
2001)
</taxonomicName>
.
</paragraph>
<paragraph id="587436C2FFB3FF9EFF5204DDFD68F98D" blockId="9.[113,764,293,1640]" pageId="9" pageNumber="10">Compared with gene knockout and knockdown, gene overexpression is highly dependent on the maintenance of introduced DNA. For species with long MAC chromosomes, the attempt can start by introducing the target gene with flanking sequences. However, for species with nano-chromosomes in the MAC, introducing the entire chromosome with telomeres is more feasible.</paragraph>
</subSubSection>
</treatment>
</document>

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<paragraph id="587436C2FFBDFF90FC79077AF94AF9A8" blockId="7.[809,1459,1586,1963]" box="[934,1335,1586,1613]" pageId="7" pageNumber="8">
<emphasis id="6ABFEAD0FFBDFF90FC79077AF8E3F9A8" bold="true" box="[934,1182,1587,1613]" pageId="7" pageNumber="8">Gene overexpression in</emphasis>
<taxonomicName id="9FCB4D41FFBDFF90FB7C077AF94AF9A8" authority="Nanney &amp; McCoy, 1976" box="[1187,1335,1586,1613]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="7" pageNumber="8" phylum="Ciliophora" rank="species" species="thermophila">
<emphasis id="6ABFEAD0FFBDFF90FB7C077AF94AF9A8" bold="true" box="[1187,1335,1586,1613]" italics="true" pageId="7" pageNumber="8">T. thermophila</emphasis>
</taxonomicName>
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<paragraph id="587436C2FFBDFF90FCF50710F85DF935" blockId="7.[809,1459,1586,1963]" pageId="7" pageNumber="8">
Overexpression is a commonly used technique in biological research and can be applied to produce economically valuable products (David-Pfeuty and
<bibRefCitation id="3C5A4B33FFBDFF90FBBB07D1F924F955" author="Nouvian-Dooghe" box="[1124,1369,1688,1712]" firstAuthor="Nouvian-Dooghe" pageId="7" pageNumber="8" pagination="3097 - 116" refId="ref9082" refString="David-Pfeuty º * Nouvian-Dooghe Y. Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. Journal of Cell Biology 1990; 111: 3097 - 116. hups: // doi. org / 10.1083 / jcb. 111.6.3097" type="journal article" year="1990">Nouvian-Dooghe 1990</bibRefCitation>
, Prelich 2012, Wang
<emphasis id="6ABFEAD0FFBDFF90FC7607F1FFAAF92A" box="[937,983,1719,1743]" italics="true" pageId="7" pageNumber="8">et al.</emphasis>
2019).
</paragraph>
<paragraph id="587436C2FFBDFF9FFC9A079EFEE4FA3B" blockId="7.[809,1459,1586,1963]" lastBlockId="8.[128,778,1259,2003]" lastPageId="8" lastPageNumber="9" pageId="7" pageNumber="8">
In
<taxonomicName id="9FCB4D41FFBDFF90FCBD0791FF89F90A" box="[866,1012,1751,1775]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="7" pageNumber="8" phylum="Ciliophora" rank="species" species="thermophila">
<emphasis id="6ABFEAD0FFBDFF90FCBD0791FF89F90A" box="[866,1012,1751,1775]" italics="true" pageId="7" pageNumber="8">T. thermophila</emphasis>
</taxonomicName>
, ribosomal DNA (rDNA) exists as a single copy in the MIC chromosome and is flanked by a chromosome breakage sequence (Yao and Gall 1977). During new MAC development in conjugation, the rDNA region is excised from the chromosome and rearranged into a 21 kb inverted repeat, termed an rDNA palindrome, then amplified to a copy number of ~10 000 (
<bibRefCitation id="3C5A4B33FFBDFF90FC4F06DAF875F84E" author="Brunk" box="[912,1032,1939,1963]" firstAuthor="Brunk" pageId="7" pageNumber="8" pagination="49 - 83" refId="ref8493" refString="Brunk CF. Genome reorganization in Tetrahymena. In: Bourne GH * Danielli JF * Jeon KW (eds.) * International Review of Cytology. London: Academic Press * 1986 * 49 - 83." type="book chapter" year="1986">Brunk 1986</bibRefCitation>
). The use of rDNA or plasmids containing rDNA replication origins as vectors to carry the target gene allows the target gene to be introduced into the MAC and maintain its high copy number (Table 1). The constructed DNA is integrated into the rDNA mini-chromosome and transcribed normally. Expression of the transformed target gene can be regulated further by modifying its promoter, such as by replacing the original promoter with the histone H4 promoter (Yao and Yao 1991, Kahn
<emphasis id="6ABFEAD0FFB2FF9FFF22048EFD51FA3B" box="[253,300,1478,1502]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
1993, Gaertig and Gorovsky 1995).
</paragraph>
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<paragraph id="587436C2FFB2FF9FFF5E02B3FD71FB5E" blockId="8.[129,1472,1018,1211]" pageId="8" pageNumber="9">
<emphasis id="6ABFEAD0FFB2FF9FFF5E02B3FCA5FBF6" bold="true" box="[129,216,1018,1043]" pageId="8" pageNumber="9">Figure 5.</emphasis>
Gene overexpression strategies in ciliates. A, construct of gene overexpression and the homologous recombination in
<taxonomicName id="9FCB4D41FFB2FF9FFAF302B2F9DEFBF6" box="[1324,1443,1019,1043]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFAF302B2F9DEFBF6" box="[1324,1443,1019,1043]" italics="true" pageId="8" pageNumber="9">Tetrahymena</emphasis>
</taxonomicName>
(a) and positive transformed cell screening (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR. Red arrows pointing to the plasmid indicate the restriction enzyme digestion sites. B, gene overexpression by injection of more copies in
<taxonomicName id="9FCB4D41FFB2FF9FFF060506FD37FB83" box="[217,330,1103,1126]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFF060506FD37FB83" box="[217,330,1103,1126]" italics="true" pageId="8" pageNumber="9">Paramecium</emphasis>
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. Telomeres are indicated with red squares at the end of chromosomes. C, gene overexpression by introduction of artificial chromosomes in
<taxonomicName id="9FCB4D41FFB2FF9FFEFC0522FD02FB66" box="[291,383,1131,1155]" class="Hypotrichea" family="Oxytrichidae" genus="Oxytricha" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFEFC0522FD02FB66" box="[291,383,1131,1155]" italics="true" pageId="8" pageNumber="9">Oxytricha</emphasis>
</taxonomicName>
. Abbreviations: GOI, gene of interest; MAC, macronucleus; MIC, micronucleus;
<emphasis id="6ABFEAD0FFB2FF9FFB5F0522F8C6FB66" box="[1152,1211,1131,1155]" italics="true" pageId="8" pageNumber="9">MMT1</emphasis>
and
<emphasis id="6ABFEAD0FFB2FF9FFB370522F95EFB66" box="[1256,1315,1131,1155]" italics="true" pageId="8" pageNumber="9">MMT3</emphasis>
, cadmiuminducible metallothionein genes;
<emphasis id="6ABFEAD0FFB2FF9FFE6305CEFD80FB7B" box="[444,509,1159,1182]" italics="true" pageId="8" pageNumber="9">neo Tet</emphasis>
, a synthetic
<emphasis id="6ABFEAD0FFB2FF9FFDAD05CEFEECFB7B" box="[626,657,1159,1182]" italics="true" pageId="8" pageNumber="9">neo</emphasis>
gene (neomycin-resistance gene) optimized for codon usage of
<taxonomicName id="9FCB4D41FFB2FF9FFB3505CEF91CFB7A" box="[1258,1377,1159,1183]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
<emphasis id="6ABFEAD0FFB2FF9FFB3505CEF91CFB7A" box="[1258,1377,1159,1183]" italics="true" pageId="8" pageNumber="9">Tetrahymena</emphasis>
</taxonomicName>
; PM, paromomycin.
</paragraph>
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<paragraph id="587436C2FFB2FF9FFF4304ACFDC0F8D2" blockId="8.[128,778,1259,2003]" pageId="8" pageNumber="9">
Another way to promote gene overexpression in
<taxonomicName id="9FCB4D41FFB2FF9FFDA404AFFF74FA18" box="[635,777,1509,1533]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="thermophila">
<emphasis id="6ABFEAD0FFB2FF9FFDA404AFFF74FA18" box="[635,777,1509,1533]" italics="true" pageId="8" pageNumber="9">T.thermophila</emphasis>
</taxonomicName>
is to use homologous recombination to replace a non-essential gene with the target gene (Table 1; Qiao
<emphasis id="6ABFEAD0FFB2FF9FFDF9076CFE28F9D9" box="[550,597,1572,1596]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
2017, Wang
<emphasis id="6ABFEAD0FFB2FF9FFD04076CFF77F9D9" box="[731,778,1572,1596]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
2019). The most frequently replaced genes are the expressioninducible genes
<emphasis id="6ABFEAD0FFB2FF9FFEF9072AFD18F99E" box="[294,357,1635,1659]" italics="true" pageId="8" pageNumber="9">MMT1</emphasis>
and
<emphasis id="6ABFEAD0FFB2FF9FFE47072AFDA5F99E" box="[408,472,1635,1659]" italics="true" pageId="8" pageNumber="9">MMT3</emphasis>
(
<figureCitation id="C0F02A47FFB2FF9FFE37072BFE4EF99F" box="[488,563,1634,1658]" captionStart="Figure 5" captionStartId="8.[129,194,1018,1042]" captionTargetBox="[138,1466,153,980]" captionTargetId="figure-518@8.[128,1475,144,991]" captionTargetPageId="8" captionText="Figure 5. Gene overexpression strategies in ciliates.A* construct of gene overexpression and the homologous recombination in Tetrahymena (a) and positive transformed cell screening (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing to the plasmid indicate the restriction enzyme digestion sites. B* gene overexpression by injection of more copies in Paramecium. ºelomeres are indicated with red squares at the end of chromosomes. C* gene overexpression by introduction of artificial chromosomes in Oxytricha. Abbreviations:GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; MT1 and MT3* cadmiuminducible metallothionein genes; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin." figureDoi="http://doi.org/10.5281/zenodo.14423119" httpUri="https://zenodo.org/record/14423119/files/figure.png" pageId="8" pageNumber="9">Fig. 5A</figureCitation>
). This is very similar to the MAC gene knockout strategy described above, except that both
<emphasis id="6ABFEAD0FFB2FF9FFF6907E8FC88F95C" box="[182,245,1697,1721]" italics="true" pageId="8" pageNumber="9">MMT1</emphasis>
and
<emphasis id="6ABFEAD0FFB2FF9FFEFA07E8FD18F95C" box="[293,357,1697,1721]" italics="true" pageId="8" pageNumber="9">MMT3</emphasis>
are replaced, with the target gene and
<emphasis id="6ABFEAD0FFB2FF9FFD3707EBFCDDF93D" italics="true" pageId="8" pageNumber="9">neo Tet</emphasis>
, respectively. It is noteworthy that the drug-resistance gene used here is only the coding region rather than the whole cassette of
<emphasis id="6ABFEAD0FFB2FF9FFF0B07B6FD7FF8F2" box="[212,258,1791,1815]" italics="true" pageId="8" pageNumber="9">neo4</emphasis>
, because expression of
<emphasis id="6ABFEAD0FFB2FF9FFE260649FE3CF8F2" box="[505,577,1792,1815]" italics="true" pageId="8" pageNumber="9">neo Tet</emphasis>
is regulated by the
<emphasis id="6ABFEAD0FFB2FF9FFF5E0656FCBDF8D2" box="[129,192,1823,1847]" italics="true" pageId="8" pageNumber="9">MMT3</emphasis>
promoter (
<figureCitation id="C0F02A47FFB2FF9FFEE90657FDFEF8D2" box="[310,387,1822,1847]" captionStart="Figure 2" captionStartId="3.[114,179,1438,1462]" captionTargetBox="[117,1455,145,1397]" captionTargetId="figure-160@3.[114,1458,144,1410]" captionTargetPageId="3" captionText="Figure 2. Gene knockout based on homologous recombination in Tetrahymena thermophila. A* schematic diagram of neo4 selectable casseưe (a) and flow path of homologous recombination (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing at the plasmid indicate the restriction enzyme digestion sites. B* cell screening of macronuclear gene knockout. A cross covering the cell means that the cell is dead. Ŋe black bars represent the endogenous chromosomes* and pink rectangles indicate the exogenous DNA that has been integrated into the chromosomes. C* procedures for micronuclear gene knockout in Tetrahymena. Ŋe correspondence between genotypes and cell icons is listed in the key at the boưom less. Abbreviations:UºR* untranslated regions; BTU2* a gene encoding β-tubulin; GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; 6-MP* 6-methylpurine; MT1* a cadmiuminducible metallothionein gene; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin." figureDoi="http://doi.org/10.5281/zenodo.14423111" httpUri="https://zenodo.org/record/14423111/files/figure.png" pageId="8" pageNumber="9">Figs 2A</figureCitation>
, 5A).
</paragraph>
<paragraph id="587436C2FFB2FF9FFF430677FEA1F836" blockId="8.[128,778,1259,2003]" pageId="8" pageNumber="9">
Replacing non-essential genes (
<emphasis id="6ABFEAD0FFB2FF9FFE2E0677FE4DF8B3" box="[497,560,1854,1878]" italics="true" pageId="8" pageNumber="9">MMT1</emphasis>
and
<emphasis id="6ABFEAD0FFB2FF9FFDB10677FED3F8B3" box="[622,686,1854,1878]" italics="true" pageId="8" pageNumber="9">MMT3</emphasis>
) with a target gene, coupled with the regulation of overexpression levels and timing by the addition and removal of Cd
<superScript id="AFBE9B8AFFB2FF9FFDBA0632FE08F86C" attach="right" box="[613,629,1915,1929]" fontSize="6" pageId="8" pageNumber="9">2+</superScript>
, offers greater control in comparison to increasing the gene copy number through rDNA-dependent replication (
<bibRefCitation id="3C5A4B33FFB2FF9FFDCE06F2FEB1F836" author="Boldrin" box="[529,716,1979,2003]" etAl="et al." firstAuthor="Boldrin" pageId="8" pageNumber="9" pagination="422 - 5" refId="ref8345" refString="Boldrin F * Santovito G * Gaertig J et al. Metallothionein gene from Tetrahymena thermophila with a copper-inducible-repressible promoter. Eukaryotic Cell 2006; 5: 422 - 5. hups: // doi. org / 10.1128 / EC. 5.2.422 - 425.2006" type="journal article" year="2006">
Boldrin
<emphasis id="6ABFEAD0FFB2FF9FFDBA06F5FEE9F836" box="[613,660,1979,2003]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
2006
</bibRefCitation>
).
</paragraph>
</subSubSection>
</treatment>
</document>