Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside
There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ...
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| Veröffentlicht in: | European journal of nuclear medicine and molecular imaging 2004-03, Vol.31 (3), p.433-438 |
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| container_title | European journal of nuclear medicine and molecular imaging |
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| creator | Lee, Kyung-Han Byun, Sang Sung Choi, Joon Hun Paik, Jin-Young Choe, Yearn Seong Kim, Byung-Tae |
| description | There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P |
| doi_str_mv | 10.1007/s00259-003-1395-7 |
| format | Article |
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The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression.</description><identifier>ISSN: 1619-7070</identifier><identifier>DOI: 10.1007/s00259-003-1395-7</identifier><identifier>PMID: 14745516</identifier><language>eng</language><publisher>Germany</publisher><subject>Animals ; BALB 3T3 Cells ; beta-Galactosidase - genetics ; beta-Galactosidase - metabolism ; Cell Line, Tumor ; Cercopithecus aethiops ; COS Cells ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Gene Expression Profiling - methods ; Gene Expression Regulation, Enzymologic - physiology ; Gene Targeting - methods ; Genes, Reporter - genetics ; Humans ; Iodine Radioisotopes - pharmacokinetics ; Lac Operon - genetics ; Metabolic Clearance Rate ; Mice ; Mice, Nude ; Neuroblastoma - diagnostic imaging ; Neuroblastoma - genetics ; Neuroblastoma - metabolism ; Radionuclide Imaging ; Radiopharmaceuticals - pharmacokinetics ; Thiogalactosides - pharmacokinetics</subject><ispartof>European journal of nuclear medicine and molecular imaging, 2004-03, Vol.31 (3), p.433-438</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14745516$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lee, Kyung-Han</creatorcontrib><creatorcontrib>Byun, Sang Sung</creatorcontrib><creatorcontrib>Choi, Joon Hun</creatorcontrib><creatorcontrib>Paik, Jin-Young</creatorcontrib><creatorcontrib>Choe, Yearn Seong</creatorcontrib><creatorcontrib>Kim, Byung-Tae</creatorcontrib><title>Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside</title><title>European journal of nuclear medicine and molecular imaging</title><addtitle>Eur J Nucl Med Mol Imaging</addtitle><description>There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression.</description><subject>Animals</subject><subject>BALB 3T3 Cells</subject><subject>beta-Galactosidase - genetics</subject><subject>beta-Galactosidase - metabolism</subject><subject>Cell Line, Tumor</subject><subject>Cercopithecus aethiops</subject><subject>COS Cells</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Gene Expression Profiling - methods</subject><subject>Gene Expression Regulation, Enzymologic - physiology</subject><subject>Gene Targeting - methods</subject><subject>Genes, Reporter - genetics</subject><subject>Humans</subject><subject>Iodine Radioisotopes - pharmacokinetics</subject><subject>Lac Operon - genetics</subject><subject>Metabolic Clearance Rate</subject><subject>Mice</subject><subject>Mice, Nude</subject><subject>Neuroblastoma - diagnostic imaging</subject><subject>Neuroblastoma - genetics</subject><subject>Neuroblastoma - metabolism</subject><subject>Radionuclide Imaging</subject><subject>Radiopharmaceuticals - pharmacokinetics</subject><subject>Thiogalactosides - pharmacokinetics</subject><issn>1619-7070</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1kL1OwzAYRT2AaCk8AAvyxGawEzu2R1TxJ1ViKQtL5MRfEqPUDrYryNsTCVjuXY6Ori5CV4zeMkrlXaK0EJpQWhJWakHkCVqzimkiqaQrdJ7SB6VMFUqfoRXjkgvBqjXyexN7yM73OHR4NO07jjCFmCHiHjxg-J4ipOSCx18uDzga64IL1nkgo2lgHMHiaQA_j5CHeSQNZEOwJXlwoTeLMYdpjsaH5CxcoNPOjAku_3qD3h4f9ttnsnt9etne70hfcJVJqyoNTHFNmaCVssoUUjalVJqLdom2KVXXFbJghZGdrVQruKS86YTUDVSm3KCbX-8Uw-cRUq4PLrXLWOMhHFMtWaV4qfQCXv-Bx-YAtp6iO5g41_8PlT8lfGhb</recordid><startdate>20040301</startdate><enddate>20040301</enddate><creator>Lee, Kyung-Han</creator><creator>Byun, Sang Sung</creator><creator>Choi, Joon Hun</creator><creator>Paik, Jin-Young</creator><creator>Choe, Yearn Seong</creator><creator>Kim, Byung-Tae</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>20040301</creationdate><title>Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside</title><author>Lee, Kyung-Han ; Byun, Sang Sung ; Choi, Joon Hun ; Paik, Jin-Young ; Choe, Yearn Seong ; Kim, Byung-Tae</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-g248t-c869e1849015068d8a277b378945c894cb38ff27212a7fd68c54704bf579be6a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Animals</topic><topic>BALB 3T3 Cells</topic><topic>beta-Galactosidase - genetics</topic><topic>beta-Galactosidase - metabolism</topic><topic>Cell Line, Tumor</topic><topic>Cercopithecus aethiops</topic><topic>COS Cells</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Gene Expression Profiling - methods</topic><topic>Gene Expression Regulation, Enzymologic - physiology</topic><topic>Gene Targeting - methods</topic><topic>Genes, Reporter - genetics</topic><topic>Humans</topic><topic>Iodine Radioisotopes - pharmacokinetics</topic><topic>Lac Operon - genetics</topic><topic>Metabolic Clearance Rate</topic><topic>Mice</topic><topic>Mice, Nude</topic><topic>Neuroblastoma - diagnostic imaging</topic><topic>Neuroblastoma - genetics</topic><topic>Neuroblastoma - metabolism</topic><topic>Radionuclide Imaging</topic><topic>Radiopharmaceuticals - pharmacokinetics</topic><topic>Thiogalactosides - pharmacokinetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lee, Kyung-Han</creatorcontrib><creatorcontrib>Byun, Sang Sung</creatorcontrib><creatorcontrib>Choi, Joon Hun</creatorcontrib><creatorcontrib>Paik, Jin-Young</creatorcontrib><creatorcontrib>Choe, Yearn Seong</creatorcontrib><creatorcontrib>Kim, Byung-Tae</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>European journal of nuclear medicine and molecular imaging</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lee, Kyung-Han</au><au>Byun, Sang Sung</au><au>Choi, Joon Hun</au><au>Paik, Jin-Young</au><au>Choe, Yearn Seong</au><au>Kim, Byung-Tae</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside</atitle><jtitle>European journal of nuclear medicine and molecular imaging</jtitle><addtitle>Eur J Nucl Med Mol Imaging</addtitle><date>2004-03-01</date><risdate>2004</risdate><volume>31</volume><issue>3</issue><spage>433</spage><epage>438</epage><pages>433-438</pages><issn>1619-7070</issn><abstract>There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression.</abstract><cop>Germany</cop><pmid>14745516</pmid><doi>10.1007/s00259-003-1395-7</doi><tpages>6</tpages></addata></record> |
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| subjects | Animals BALB 3T3 Cells beta-Galactosidase - genetics beta-Galactosidase - metabolism Cell Line, Tumor Cercopithecus aethiops COS Cells Escherichia coli - enzymology Escherichia coli - genetics Gene Expression Profiling - methods Gene Expression Regulation, Enzymologic - physiology Gene Targeting - methods Genes, Reporter - genetics Humans Iodine Radioisotopes - pharmacokinetics Lac Operon - genetics Metabolic Clearance Rate Mice Mice, Nude Neuroblastoma - diagnostic imaging Neuroblastoma - genetics Neuroblastoma - metabolism Radionuclide Imaging Radiopharmaceuticals - pharmacokinetics Thiogalactosides - pharmacokinetics |
| title | Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside |
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