Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines
Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are...
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| description | Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes. |
| doi_str_mv | 10.1371/journal.pone.0221034 |
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More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0221034</identifier><identifier>PMID: 31430311</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Allergies ; Animals ; Biology and Life Sciences ; Biotechnology ; Calibration ; Cell Line, Tumor ; Cell surface ; Chains ; Copy number ; Diagnostic software ; Diagnostic systems ; Endoplasmic reticulum ; Flow cytometry ; Gene Dosage ; Gene expression ; Gene Expression Profiling ; Genes ; Genes, Immunoglobulin Heavy Chain - genetics ; Genes, Reporter - genetics ; Immunoglobulin E ; Immunoglobulin gamma-Chains - genetics ; Immunoglobulin gamma-Chains - metabolism ; Immunoglobulins ; Leukemia ; Leukemia, Basophilic, Acute - genetics ; Leukemia, Basophilic, Acute - pathology ; Microspheres ; Monoclonal antibodies ; mRNA ; NF-AT protein ; Parameter sensitivity ; Pharmacy ; Rats ; Receptors ; Receptors, IgE - genetics ; Receptors, IgE - metabolism ; Research and Analysis Methods ; Sensitivity ; Transfection ; Tumor cell lines</subject><ispartof>PloS one, 2019-08, Vol.14 (8), p.e0221034-e0221034</ispartof><rights>2019 Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Ali et al 2019 Ali et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-f8f7f78983cf6424ff64102767482c966e396816f33d1f841b5bc3064400ee8f3</citedby><cites>FETCH-LOGICAL-c526t-f8f7f78983cf6424ff64102767482c966e396816f33d1f841b5bc3064400ee8f3</cites><orcidid>0000-0002-7491-3414 ; 0000-0002-1732-9932 ; 0000-0002-7756-3798</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701790/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701790/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31430311$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ali, Eman Ali</creatorcontrib><creatorcontrib>Kalli, Marina</creatorcontrib><creatorcontrib>Wan, Daniel</creatorcontrib><creatorcontrib>Nakamura, Ryosuke</creatorcontrib><creatorcontrib>Onion, David</creatorcontrib><creatorcontrib>Alanine, Daniel G W</creatorcontrib><creatorcontrib>Alcocer, Marcos J C</creatorcontrib><creatorcontrib>Falcone, Franco H</creatorcontrib><title>Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.</description><subject>Allergies</subject><subject>Animals</subject><subject>Biology and Life Sciences</subject><subject>Biotechnology</subject><subject>Calibration</subject><subject>Cell Line, Tumor</subject><subject>Cell surface</subject><subject>Chains</subject><subject>Copy number</subject><subject>Diagnostic software</subject><subject>Diagnostic systems</subject><subject>Endoplasmic reticulum</subject><subject>Flow cytometry</subject><subject>Gene Dosage</subject><subject>Gene expression</subject><subject>Gene Expression Profiling</subject><subject>Genes</subject><subject>Genes, Immunoglobulin Heavy Chain - genetics</subject><subject>Genes, Reporter - genetics</subject><subject>Immunoglobulin E</subject><subject>Immunoglobulin gamma-Chains - genetics</subject><subject>Immunoglobulin gamma-Chains - metabolism</subject><subject>Immunoglobulins</subject><subject>Leukemia</subject><subject>Leukemia, Basophilic, Acute - genetics</subject><subject>Leukemia, Basophilic, Acute - pathology</subject><subject>Microspheres</subject><subject>Monoclonal antibodies</subject><subject>mRNA</subject><subject>NF-AT protein</subject><subject>Parameter sensitivity</subject><subject>Pharmacy</subject><subject>Rats</subject><subject>Receptors</subject><subject>Receptors, IgE - genetics</subject><subject>Receptors, IgE - metabolism</subject><subject>Research and Analysis Methods</subject><subject>Sensitivity</subject><subject>Transfection</subject><subject>Tumor cell lines</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNptUs1u1DAQjhCIlsIbILDEpRx2sWPH8V6QYEVhpZWQKjhbE2e86yWxg50g2ifgdTjwGn0mku62ahGXsWV_PzOjL8ueMzpnvGRvdmGIHpp5FzzOaZ4zysWD7JgteD6TOeUP79yPsicp7SgtuJLycXbEmeCUM3ac_VpuIYLpMbpL6F3wJFiyHVrw5Mxc_TlfXf0mZgvOE_zZRUxpgoCvyQY9EhO6C-KHtsJIRsg1z11iTSL0pIIUuq1rnCENDt8AWwfk9Pz9-jWJ2IU4ehKDTUMa5zE9zR5ZaBI-O5wn2dezD1-Wn2brzx9Xy3frmSly2c-ssqUt1UJxY6XIhR0ro3kpS6Fys5AS-UIqJi3nNbNKsKqoDKdSCEoRleUn2cu9bteEpA9LTDofJRQfCx0Rqz2iDrDTXXQtxAsdwOnrhxA3GmLvTIO6ULIGkSNIUQlFoZK1ZXWtpMlrCWJye3twG6oWa4O-j9DcE73_491Wb8IPLUvKysXUzOlBIIbvA6Zety5NWwOPYZj6VgWjrGDlCH31D_T_04k9ysSQUkR72wyjegrWDUtPwdKHYI20F3cHuSXdJIn_BV01znA</recordid><startdate>20190820</startdate><enddate>20190820</enddate><creator>Ali, Eman Ali</creator><creator>Kalli, Marina</creator><creator>Wan, Daniel</creator><creator>Nakamura, Ryosuke</creator><creator>Onion, David</creator><creator>Alanine, Daniel G W</creator><creator>Alcocer, Marcos J C</creator><creator>Falcone, Franco H</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-7491-3414</orcidid><orcidid>https://orcid.org/0000-0002-1732-9932</orcidid><orcidid>https://orcid.org/0000-0002-7756-3798</orcidid></search><sort><creationdate>20190820</creationdate><title>Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines</title><author>Ali, Eman Ali ; Kalli, Marina ; Wan, Daniel ; Nakamura, Ryosuke ; Onion, David ; Alanine, Daniel G W ; Alcocer, Marcos J C ; Falcone, Franco H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c526t-f8f7f78983cf6424ff64102767482c966e396816f33d1f841b5bc3064400ee8f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Allergies</topic><topic>Animals</topic><topic>Biology and Life Sciences</topic><topic>Biotechnology</topic><topic>Calibration</topic><topic>Cell Line, Tumor</topic><topic>Cell surface</topic><topic>Chains</topic><topic>Copy number</topic><topic>Diagnostic software</topic><topic>Diagnostic systems</topic><topic>Endoplasmic reticulum</topic><topic>Flow cytometry</topic><topic>Gene Dosage</topic><topic>Gene expression</topic><topic>Gene Expression Profiling</topic><topic>Genes</topic><topic>Genes, Immunoglobulin Heavy Chain - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ali, Eman Ali</au><au>Kalli, Marina</au><au>Wan, Daniel</au><au>Nakamura, Ryosuke</au><au>Onion, David</au><au>Alanine, Daniel G W</au><au>Alcocer, Marcos J C</au><au>Falcone, Franco H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2019-08-20</date><risdate>2019</risdate><volume>14</volume><issue>8</issue><spage>e0221034</spage><epage>e0221034</epage><pages>e0221034-e0221034</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31430311</pmid><doi>10.1371/journal.pone.0221034</doi><orcidid>https://orcid.org/0000-0002-7491-3414</orcidid><orcidid>https://orcid.org/0000-0002-1732-9932</orcidid><orcidid>https://orcid.org/0000-0002-7756-3798</orcidid><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_2276832760 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Allergies Animals Biology and Life Sciences Biotechnology Calibration Cell Line, Tumor Cell surface Chains Copy number Diagnostic software Diagnostic systems Endoplasmic reticulum Flow cytometry Gene Dosage Gene expression Gene Expression Profiling Genes Genes, Immunoglobulin Heavy Chain - genetics Genes, Reporter - genetics Immunoglobulin E Immunoglobulin gamma-Chains - genetics Immunoglobulin gamma-Chains - metabolism Immunoglobulins Leukemia Leukemia, Basophilic, Acute - genetics Leukemia, Basophilic, Acute - pathology Microspheres Monoclonal antibodies mRNA NF-AT protein Parameter sensitivity Pharmacy Rats Receptors Receptors, IgE - genetics Receptors, IgE - metabolism Research and Analysis Methods Sensitivity Transfection Tumor cell lines |
| title | Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-04T00%3A10%3A12IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Characterization%20of%20human%20Fc%CE%B5RI%CE%B1%20chain%20expression%20and%20gene%20copy%20number%20in%20humanized%20rat%20basophilic%20leukaemia%20(RBL)%20reporter%20cell%20lines&rft.jtitle=PloS%20one&rft.au=Ali,%20Eman%20Ali&rft.date=2019-08-20&rft.volume=14&rft.issue=8&rft.spage=e0221034&rft.epage=e0221034&rft.pages=e0221034-e0221034&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0221034&rft_dat=%3Cproquest_plos_%3E2285101517%3C/proquest_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2276832760&rft_id=info:pmid/31430311&rft_doaj_id=oai_doaj_org_article_586da42ea64b480ab6df1dd86c2d6a4f&rfr_iscdi=true |