Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage
Identifieur interne : 002084 ( Istex/Corpus ); précédent : 002083; suivant : 002085Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage
Auteurs : Lorena Urbanelli ; Chiara Ronchini ; Laura Fontana ; Sylvie Menard ; Rosaria Orlandi ; Paolo MonaciSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 2001.
English descriptors
- KwdEn :
- Teeft :
- Assay, Average values, Biol, Capsid, Clone, Elisa, Gene, Gene delivery, Gene delivery vehicles, Gene expression, Gene transduction, Gene transfer, Her2, Independent assays, Lamentous, Lamentous phage, Ligand, Luciferase, Luciferase activity, Luciferase assay, Luciferase expression, Magnetic beads, Mammalian cells, Mewo, Minutes incubation, Monaci, Pbbs, Peptide, Phage, Phage display, Phage particles, Phage vectors, Phagemid, Piii, Plasmid, Pviii, Pviii proteins, Random peptide library, Receptor, Room temperature, Skbr3, Skbr3 cells, Standard deviation, Transduction, Transfection, Triplicate determinations, Unbound, Unbound phage.
Abstract
Abstract: Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.
Url:
DOI: 10.1006/jmbi.2001.5111
Links to Exploration step
ISTEX:1E7ADEACE591D8C14BF36A74093423AAF93C7BC6Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title><author><name sortKey="Urbanelli, Lorena" sort="Urbanelli, Lorena" uniqKey="Urbanelli L" first="Lorena" last="Urbanelli">Lorena Urbanelli</name><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author><author><name sortKey="Ronchini, Chiara" sort="Ronchini, Chiara" uniqKey="Ronchini C" first="Chiara" last="Ronchini">Chiara Ronchini</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Fontana, Laura" sort="Fontana, Laura" uniqKey="Fontana L" first="Laura" last="Fontana">Laura Fontana</name><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author><author><name sortKey="Menard, Sylvie" sort="Menard, Sylvie" uniqKey="Menard S" first="Sylvie" last="Menard">Sylvie Menard</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Orlandi, Rosaria" sort="Orlandi, Rosaria" uniqKey="Orlandi R" first="Rosaria" last="Orlandi">Rosaria Orlandi</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Monaci, Paolo" sort="Monaci, Paolo" uniqKey="Monaci P" first="Paolo" last="Monaci">Paolo Monaci</name><affiliation><mods:affiliation>E-mail: paolo_monaci@merck.com</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:1E7ADEACE591D8C14BF36A74093423AAF93C7BC6</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1006/jmbi.2001.5111</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002084</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002084</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title><author><name sortKey="Urbanelli, Lorena" sort="Urbanelli, Lorena" uniqKey="Urbanelli L" first="Lorena" last="Urbanelli">Lorena Urbanelli</name><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author><author><name sortKey="Ronchini, Chiara" sort="Ronchini, Chiara" uniqKey="Ronchini C" first="Chiara" last="Ronchini">Chiara Ronchini</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Fontana, Laura" sort="Fontana, Laura" uniqKey="Fontana L" first="Laura" last="Fontana">Laura Fontana</name><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author><author><name sortKey="Menard, Sylvie" sort="Menard, Sylvie" uniqKey="Menard S" first="Sylvie" last="Menard">Sylvie Menard</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Orlandi, Rosaria" sort="Orlandi, Rosaria" uniqKey="Orlandi R" first="Rosaria" last="Orlandi">Rosaria Orlandi</name><affiliation><mods:affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</mods:affiliation></affiliation></author><author><name sortKey="Monaci, Paolo" sort="Monaci, Paolo" uniqKey="Monaci P" first="Paolo" last="Monaci">Paolo Monaci</name><affiliation><mods:affiliation>E-mail: paolo_monaci@merck.com</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001">2001</date><biblScope unit="volume">313</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="965">965</biblScope><biblScope unit="page" to="976">976</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>AP</term><term>BSA</term><term>CAR</term><term>CMV</term><term>ECD</term><term>FACS</term><term>FCS</term><term>HER2</term><term>HER2/neu receptor</term><term>HRP</term><term>RLU</term><term>TU</term><term>cancer</term><term>dsDNA</term><term>gene delivery</term><term>mAb</term><term>phage display</term><term>ssDNA</term><term>targeting</term><term>wt</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Assay</term><term>Average values</term><term>Biol</term><term>Capsid</term><term>Clone</term><term>Elisa</term><term>Gene</term><term>Gene delivery</term><term>Gene delivery vehicles</term><term>Gene expression</term><term>Gene transduction</term><term>Gene transfer</term><term>Her2</term><term>Independent assays</term><term>Lamentous</term><term>Lamentous phage</term><term>Ligand</term><term>Luciferase</term><term>Luciferase activity</term><term>Luciferase assay</term><term>Luciferase expression</term><term>Magnetic beads</term><term>Mammalian cells</term><term>Mewo</term><term>Minutes incubation</term><term>Monaci</term><term>Pbbs</term><term>Peptide</term><term>Phage</term><term>Phage display</term><term>Phage particles</term><term>Phage vectors</term><term>Phagemid</term><term>Piii</term><term>Plasmid</term><term>Pviii</term><term>Pviii proteins</term><term>Random peptide library</term><term>Receptor</term><term>Room temperature</term><term>Skbr3</term><term>Skbr3 cells</term><term>Standard deviation</term><term>Transduction</term><term>Transfection</term><term>Triplicate determinations</term><term>Unbound</term><term>Unbound phage</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>phage</json:string><json:string>her2</json:string><json:string>skbr3</json:string><json:string>receptor</json:string><json:string>clone</json:string><json:string>luciferase</json:string><json:string>pviii</json:string><json:string>peptide</json:string><json:string>transduction</json:string><json:string>skbr3 cells</json:string><json:string>lamentous</json:string><json:string>elisa</json:string><json:string>ligand</json:string><json:string>gene delivery</json:string><json:string>piii</json:string><json:string>plasmid</json:string><json:string>biol</json:string><json:string>mewo</json:string><json:string>transfection</json:string><json:string>monaci</json:string><json:string>gene transduction</json:string><json:string>luciferase activity</json:string><json:string>pbbs</json:string><json:string>capsid</json:string><json:string>mammalian cells</json:string><json:string>phagemid</json:string><json:string>average values</json:string><json:string>phage particles</json:string><json:string>room temperature</json:string><json:string>unbound phage</json:string><json:string>luciferase expression</json:string><json:string>independent assays</json:string><json:string>assay</json:string><json:string>luciferase assay</json:string><json:string>gene expression</json:string><json:string>minutes incubation</json:string><json:string>gene transfer</json:string><json:string>lamentous phage</json:string><json:string>magnetic beads</json:string><json:string>phage display</json:string><json:string>pviii proteins</json:string><json:string>phage vectors</json:string><json:string>random peptide library</json:string><json:string>triplicate determinations</json:string><json:string>standard deviation</json:string><json:string>gene delivery vehicles</json:string><json:string>unbound</json:string><json:string>gene</json:string><json:string>reporter gene</json:string><json:string>phage supernatants</json:string><json:string>human epidermal growth factor receptor</json:string><json:string>soluble receptor</json:string><json:string>monoclonal antibodies</json:string><json:string>phage library</json:string><json:string>target cells</json:string><json:string>elisa results</json:string><json:string>target receptor</json:string><json:string>monoclonal antibody</json:string><json:string>cell lines</json:string><json:string>foreign peptides</json:string><json:string>extracellular domain</json:string><json:string>foreign peptide sequence</json:string><json:string>negative control</json:string><json:string>different her2 levels</json:string><json:string>elisa reader</json:string><json:string>labsystems multiskan bichromatic</json:string><json:string>elisa data</json:string><json:string>lamentous bacteriophage</json:string></teeft></keywords><author><json:item><name>Lorena Urbanelli</name><affiliations><json:string>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</json:string></affiliations></json:item><json:item><name>Chiara Ronchini</name><affiliations><json:string>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</json:string></affiliations></json:item><json:item><name>Laura Fontana</name><affiliations><json:string>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</json:string></affiliations></json:item><json:item><name>Sylvie Menard</name><affiliations><json:string>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</json:string></affiliations></json:item><json:item><name>Rosaria Orlandi</name><affiliations><json:string>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</json:string></affiliations></json:item><json:item><name>Paolo Monaci</name><affiliations><json:string>E-mail: paolo_monaci@merck.com</json:string><json:string>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>phage display</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>targeting</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HER2/neu receptor</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>gene delivery</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>cancer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AP : alkaline phosphatase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BSA : bovine serum albumin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CAR : Coxsackie-adenovirus receptor</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CMV : human cytomegalovirus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>dsDNA : double-stranded DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ECD : extracellular domain</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FACS : fluorescence-activated cell sorting</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FCS : fetal calf serum</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HER2 : human epidermal growth factor receptor 2</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HRP : horseradish peroxidase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>mAb : monoclonal antibody</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RLU : relative light units</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ssDNA : single-stranded DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TU : transducing units</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>wt : wild-type</value></json:item></subject><arkIstex>ark:/67375/6H6-T9090RX7-L</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.</abstract><qualityIndicators><score>8.776</score><pdfWordCount>6611</pdfWordCount><pdfCharCount>41128</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>595 x 842 pts (A4)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>148</abstractWordCount><abstractCharCount>1011</abstractCharCount><keywordCount>21</keywordCount></qualityIndicators><title>Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title><pmid><json:string>11700053</json:string></pmid><pii><json:string>S0022-2836(01)95111-3</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>2001</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0259-8</json:string></pii><volume>313</volume><issue>5</issue><pages><first>965</first><last>976</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2001</json:string><json:string>1999</json:string></date><geogName></geogName><orgName><json:string>European Institute of Oncology, Milano, Italy</json:string><json:string>Weizmann Institute of Science, Rehovot, Israel</json:string><json:string>Gene Therapy Systems, Inc., San Diego</json:string><json:string>American Type Culture Collection</json:string><json:string>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy Screening</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Y. Yarden</json:string><json:string>Paolo Monaci</json:string><json:string>Lorena Urbanelli</json:string><json:string>Sylvie Menard</json:string><json:string>Laura Fontana</json:string><json:string>ELISA Cells</json:string><json:string>P. Di Fiore</json:string><json:string>Data</json:string><json:string>Larocca</json:string><json:string>J. Clench</json:string><json:string>O. Minenkova</json:string><json:string>La Jolla</json:string><json:string>Chiara Ronchini</json:string><json:string>I.R.B.M. P. Angeletti</json:string></persName><placeName><json:string>Uppsala</json:string><json:string>Finland</json:string><json:string>Norway</json:string><json:string>Oslo</json:string><json:string>Helsinki</json:string><json:string>Denmark</json:string><json:string>Italy</json:string><json:string>Sweden</json:string><json:string>Roskilde</json:string></placeName><ref_url><json:string>http://www.idealibrary.com</json:string></ref_url><ref_bibl><json:string>Neuner et al.</json:string><json:string>Monaci et al.</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-T9090RX7-L</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - immunologie fondamentale</json:string></inist></categories><publicationDate>2001</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1006/jmbi.2001.5111</json:string></doi><id>1E7ADEACE591D8C14BF36A74093423AAF93C7BC6</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2001 Academic Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2001</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note>Edited by J. Karn</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Characterisation of clones derived from the screening of phage-displayed libraries. (a) Amino acid sequences of HER2-binding clones. The amino acid sequences of clones derived from the screening of the pVIII-12aa random peptide library (NL1 and NL2) and of the pVIII-NL1 “secondary” library (NL1.1 to NL1.5) are reported in the single-letter code. Sequences of foreign epitopes are indicated with grey boxes. pVIII sequences flanking the foreign epitopes are reported. Bold characters indicate residues conserved among the different clones. (b) Binding of selected clones to HER2-ECD. Phage supernatants of clones derived from the screening of pVIII-12aa or pVIII-NL1 library were tested by phage-ELISA for their binding to immobilised HER-ECD. Wild-type phage, which does not display recombinant pVIII proteins on its surface, was included as negative control. White and black bars indicate binding in the absence or in the presence of HER-ECD, respectively. ELISA results are expressed as A=A450 nm−A620 nm and are average values from two independent assays. The range for each value is reported</note><note type="content">Figure 2: Binding of HER2-ECD to phage NL1 and NL1.1. Binding of soluble HER2-ECD (5 μg/ml) to immobilised phage NL1 or NL1.1 was measured by ELISA. Wild-type phage, which does not display recombinant pVIII proteins on its surface, was included as negative control. ELISA results are expressed as A=A405 nm−A620 nm and are average values from two independent assays. The range for each value is reported.</note><note type="content">Figure 3: Characterisation of clones NL1 and NL1.1 binding to various cell lines expressing different HER2 levels. Binding of wt, NL1 and NL1.1 phage to MeWo, SKBr3, NIH3T3 and NIH3T3/neu cells was measured by flow cytometric analysis using anti-M13 antibody as described in Materials and Methods. SKBr3 cells display much higher levels of HER2 receptors on their surface as compared to MeWo cells. Similarly, NIH3T3/neu cells highly express human HER2 as compared to the parental mouse NIH3T3 cell line. Positive staining with each phage (black) is seen relative to the absence of phage (white). A representative of three separate experiments is shown.</note><note type="content">Figure 4: Binding of pVIII and pIII-displayed NL1 and NL1.1 peptides to HER2-ECD. Phage supernatants of clones displaying the NL1 or the NL1.1 peptide sequence fused to the N terminus of pVIII (NL1 and NL1.1) or pIII (pIII-NL1 and pIII-NL1.1) were tested by phage-ELISA for their binding to immobilised HER-ECD. Parental phage (pC89 and fK1), which do not display recombinant proteins on their surface, were included as negative control (wt). Phage displaying the AHNP peptide sequence as an N-terminal fusion to pIII (pIII-AHNP) or pVIII (pVIII-AHNP) were tested similarly12. Black and white bars indicate binding of pVIII and pIII-displayed peptides, respectively. ELISA results are expressed as A=A450 nm−A620 nm and are average values from two independent assays. The range for each value is reported.</note><note type="content">Figure 5: NL1.1-luc-mediated gene transfer to cell lines expressing different HER2 levels. Increasing amounts of NL1.1-luc or wt-luc phage were incubated with each cell type for four hours, then unbound phage were removed, cells washed and incubated for 48 hours before luciferase assay. Gene transfer index (y axis) is defined as the ratio between the mean luciferase activity for cells incubated with NL1.1-luc phage to that for cells incubated with an equal amount of wt-luc phage. The data were obtained using SKBr3 (indicated with rhombuses), NIH3T3/neu (triangles), MeWo (circles) and NIH3T3 (squares) cells. The number of phage particles is reported on the abscissa. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported.</note><note type="content">Figure 6: Characterisation of NL1.1-luc gene delivery to SKBr3 cells. (a) Inhibition of NL1.1-luc binding by HER2-ECD. Binding of NL1.1-luc phage to HER2-positive SKBr3 cells was measured by phage-ELISA in the presence of increasing concentrations of HER2-ECD. The concentrations of soluble receptor are indicated on the abscissa, while the percentage of binding as compared to that obtained in the absence of HER2-ECD is reported on the y axis. Average values from two independent experiments are reported. The broken line indicates the binding of wt phage. (b) Inhibition of NL1.1-luc gene transduction by HER2-ECD. NL1.1-luc phage (5×109 TU) was incubated with HER2-positive SKBr3 cells for four hours in the absence (white bar) or in the presence (black bar) of 10μg/ml of HER2-ECD. Then unbound phage were removed, and the cells were washed and incubated for 48 hours before luciferase assay. Data are expressed as percentage of luciferase activity obtained in the absence of soluble receptor. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported. (c) Inhibition of binding of NL1.1-luc by anti-HER2 mAb. Binding of NL1.1 phage (4×1010 TU) to HER2-positive SKBr3 cells was measured by phage-ELISA in the absence (white bar), or in the presence of 10 mg/ml of an unrelated mAb, mAb.MGr2 or mAb.MGr611. Data are expressed as percentage of binding as compared to that obtained in the absence of any mAb. Average values (and the range) from two independent experiments are reported. (d) Inhibition of NL1.1-luc gene transduction by anti-HER2 mAb. NL1.1-luc phage (4×1010 TU) was incubated with HER2-positive SKBr3 cells for four hours in the absence (white bar) or in the presence of 10 mg/ml of an unrelated mAb, mAb.MGr2 or mAb.MGr6. Then unbound phage were removed, the cells were washed and incubated for 48 hours before luciferase assay. Data are expressed as percentage of luciferase activity obtained in the absence of any mAb. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported.</note><note type="content">Figure 7: Effect of incubation time on NL1.1-luc transfection efficiency. A total of 5×1010 TU of NL1.1-luc (filled circles) or wt-luc (empty circles) phage was incubated at 37°C with SKBr3 cells for increasing times. Then unbound phage were removed, the cells were washed and incubated for 48 hours before luciferase assay. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported. Luciferase activity in mock-transfected controls was 2.4×104 (±1.7×103) RLU/mg protein.</note><note type="content">Table 1: Transfection efficiency in SKBr3 cells</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title><author xml:id="author-0000"><persName><forename type="first">Lorena</forename><surname>Urbanelli</surname></persName><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Chiara</forename><surname>Ronchini</surname></persName><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Laura</forename><surname>Fontana</surname></persName><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Sylvie</forename><surname>Menard</surname></persName><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Rosaria</forename><surname>Orlandi</surname></persName><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Paolo</forename><surname>Monaci</surname></persName><email>paolo_monaci@merck.com</email><note type="correspondence"><p>Corresponding author</p></note><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation></author><idno type="istex">1E7ADEACE591D8C14BF36A74093423AAF93C7BC6</idno><idno type="ark">ark:/67375/6H6-T9090RX7-L</idno><idno type="DOI">10.1006/jmbi.2001.5111</idno><idno type="PII">S0022-2836(01)95111-3</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0259-8</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001"></date><biblScope unit="volume">313</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="965">965</biblScope><biblScope unit="page" to="976">976</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>phage display</term></item><item><term>targeting</term></item><item><term>HER2/neu receptor</term></item><item><term>gene delivery</term></item><item><term>cancer</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>AP</term><term>alkaline phosphatase</term></item><item><term>BSA</term><term>bovine serum albumin</term></item><item><term>CAR</term><term>Coxsackie-adenovirus receptor</term></item><item><term>CMV</term><term>human cytomegalovirus</term></item><item><term>dsDNA</term><term>double-stranded DNA</term></item><item><term>ECD</term><term>extracellular domain</term></item><item><term>FACS</term><term>fluorescence-activated cell sorting</term></item><item><term>FCS</term><term>fetal calf serum</term></item><item><term>HER2</term><term>human epidermal growth factor receptor 2</term></item><item><term>HRP</term><term>horseradish peroxidase</term></item><item><term>mAb</term><term>monoclonal antibody</term></item><item><term>RLU</term><term>relative light units</term></item><item><term>ssDNA</term><term>single-stranded DNA</term></item><item><term>TU</term><term>transducing units</term></item><item><term>wt</term><term>wild-type</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2001-08-31">Modified</change><change when="2001">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="GR5"></istex:entity><istex:entity SYSTEM="gr6a" NDATA="IMAGE" name="GR6a"></istex:entity><istex:entity SYSTEM="gr6b" NDATA="IMAGE" name="GR6b"></istex:entity><istex:entity SYSTEM="gr6c" NDATA="IMAGE" name="GR6c"></istex:entity><istex:entity SYSTEM="gr6d" NDATA="IMAGE" name="GR6d"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>95111</aid><ce:pii>S0022-2836(01)95111-3</ce:pii><ce:doi>10.1006/jmbi.2001.5111</ce:doi><ce:copyright type="full-transfer" year="2001">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by J. Karn</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:given-name>Lorena</ce:given-name><ce:surname>Urbanelli</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Chiara</ce:given-name><ce:surname>Ronchini</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Laura</ce:given-name><ce:surname>Fontana</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Sylvie</ce:given-name><ce:surname>Menard</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Rosaria</ce:given-name><ce:surname>Orlandi</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Paolo</ce:given-name><ce:surname>Monaci</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>paolo_monaci@merck.com</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence></ce:author-group><ce:date-received day="9" month="7" year="2001"></ce:date-received><ce:date-revised day="31" month="8" year="2001"></ce:date-revised><ce:date-accepted day="25" month="9" year="2001"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>phage display</ce:text></ce:keyword><ce:keyword><ce:text>targeting</ce:text></ce:keyword><ce:keyword><ce:text>HER2/neu receptor</ce:text></ce:keyword><ce:keyword><ce:text>gene delivery</ce:text></ce:keyword><ce:keyword><ce:text>cancer</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>AP</ce:text><ce:keyword><ce:text>alkaline phosphatase</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>BSA</ce:text><ce:keyword><ce:text>bovine serum albumin</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>CAR</ce:text><ce:keyword><ce:text>Coxsackie-adenovirus receptor</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>CMV</ce:text><ce:keyword><ce:text>human cytomegalovirus</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>dsDNA</ce:text><ce:keyword><ce:text>double-stranded DNA</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ECD</ce:text><ce:keyword><ce:text>extracellular domain</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>FACS</ce:text><ce:keyword><ce:text>fluorescence-activated cell sorting</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>FCS</ce:text><ce:keyword><ce:text>fetal calf serum</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>HER2</ce:text><ce:keyword><ce:text>human epidermal growth factor receptor 2</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>HRP</ce:text><ce:keyword><ce:text>horseradish peroxidase</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>mAb</ce:text><ce:keyword><ce:text>monoclonal antibody</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>RLU</ce:text><ce:keyword><ce:text>relative light units</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ssDNA</ce:text><ce:keyword><ce:text>single-stranded DNA</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>TU</ce:text><ce:keyword><ce:text>transducing units</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>wt</ce:text><ce:keyword><ce:text>wild-type</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage</title></titleInfo><name type="personal"><namePart type="given">Lorena</namePart><namePart type="family">Urbanelli</namePart><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Chiara</namePart><namePart type="family">Ronchini</namePart><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Laura</namePart><namePart type="family">Fontana</namePart><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Sylvie</namePart><namePart type="family">Menard</namePart><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Rosaria</namePart><namePart type="family">Orlandi</namePart><affiliation>Department of Experimental Oncology, Molecular Target Unit, Istituto Nazionale Tumori, Milano, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Paolo</namePart><namePart type="family">Monaci</namePart><affiliation>E-mail: paolo_monaci@merck.com</affiliation><affiliation>Department of Molecular & Cell Biology, I.R.B.M. P. Angeletti, Pomezia (Roma) Italy</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued><dateModified encoding="w3cdtf">2001-08-31</dateModified><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.</abstract><note type="footnote">Edited by J. Karn</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Characterisation of clones derived from the screening of phage-displayed libraries. (a) Amino acid sequences of HER2-binding clones. The amino acid sequences of clones derived from the screening of the pVIII-12aa random peptide library (NL1 and NL2) and of the pVIII-NL1 “secondary” library (NL1.1 to NL1.5) are reported in the single-letter code. Sequences of foreign epitopes are indicated with grey boxes. pVIII sequences flanking the foreign epitopes are reported. Bold characters indicate residues conserved among the different clones. (b) Binding of selected clones to HER2-ECD. Phage supernatants of clones derived from the screening of pVIII-12aa or pVIII-NL1 library were tested by phage-ELISA for their binding to immobilised HER-ECD. Wild-type phage, which does not display recombinant pVIII proteins on its surface, was included as negative control. White and black bars indicate binding in the absence or in the presence of HER-ECD, respectively. ELISA results are expressed as A=A450 nm−A620 nm and are average values from two independent assays. The range for each value is reported</note><note type="content">Figure 2: Binding of HER2-ECD to phage NL1 and NL1.1. Binding of soluble HER2-ECD (5 μg/ml) to immobilised phage NL1 or NL1.1 was measured by ELISA. Wild-type phage, which does not display recombinant pVIII proteins on its surface, was included as negative control. ELISA results are expressed as A=A405 nm−A620 nm and are average values from two independent assays. The range for each value is reported.</note><note type="content">Figure 3: Characterisation of clones NL1 and NL1.1 binding to various cell lines expressing different HER2 levels. Binding of wt, NL1 and NL1.1 phage to MeWo, SKBr3, NIH3T3 and NIH3T3/neu cells was measured by flow cytometric analysis using anti-M13 antibody as described in Materials and Methods. SKBr3 cells display much higher levels of HER2 receptors on their surface as compared to MeWo cells. Similarly, NIH3T3/neu cells highly express human HER2 as compared to the parental mouse NIH3T3 cell line. Positive staining with each phage (black) is seen relative to the absence of phage (white). A representative of three separate experiments is shown.</note><note type="content">Figure 4: Binding of pVIII and pIII-displayed NL1 and NL1.1 peptides to HER2-ECD. Phage supernatants of clones displaying the NL1 or the NL1.1 peptide sequence fused to the N terminus of pVIII (NL1 and NL1.1) or pIII (pIII-NL1 and pIII-NL1.1) were tested by phage-ELISA for their binding to immobilised HER-ECD. Parental phage (pC89 and fK1), which do not display recombinant proteins on their surface, were included as negative control (wt). Phage displaying the AHNP peptide sequence as an N-terminal fusion to pIII (pIII-AHNP) or pVIII (pVIII-AHNP) were tested similarly12. Black and white bars indicate binding of pVIII and pIII-displayed peptides, respectively. ELISA results are expressed as A=A450 nm−A620 nm and are average values from two independent assays. The range for each value is reported.</note><note type="content">Figure 5: NL1.1-luc-mediated gene transfer to cell lines expressing different HER2 levels. Increasing amounts of NL1.1-luc or wt-luc phage were incubated with each cell type for four hours, then unbound phage were removed, cells washed and incubated for 48 hours before luciferase assay. Gene transfer index (y axis) is defined as the ratio between the mean luciferase activity for cells incubated with NL1.1-luc phage to that for cells incubated with an equal amount of wt-luc phage. The data were obtained using SKBr3 (indicated with rhombuses), NIH3T3/neu (triangles), MeWo (circles) and NIH3T3 (squares) cells. The number of phage particles is reported on the abscissa. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported.</note><note type="content">Figure 6: Characterisation of NL1.1-luc gene delivery to SKBr3 cells. (a) Inhibition of NL1.1-luc binding by HER2-ECD. Binding of NL1.1-luc phage to HER2-positive SKBr3 cells was measured by phage-ELISA in the presence of increasing concentrations of HER2-ECD. The concentrations of soluble receptor are indicated on the abscissa, while the percentage of binding as compared to that obtained in the absence of HER2-ECD is reported on the y axis. Average values from two independent experiments are reported. The broken line indicates the binding of wt phage. (b) Inhibition of NL1.1-luc gene transduction by HER2-ECD. NL1.1-luc phage (5×109 TU) was incubated with HER2-positive SKBr3 cells for four hours in the absence (white bar) or in the presence (black bar) of 10μg/ml of HER2-ECD. Then unbound phage were removed, and the cells were washed and incubated for 48 hours before luciferase assay. Data are expressed as percentage of luciferase activity obtained in the absence of soluble receptor. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported. (c) Inhibition of binding of NL1.1-luc by anti-HER2 mAb. Binding of NL1.1 phage (4×1010 TU) to HER2-positive SKBr3 cells was measured by phage-ELISA in the absence (white bar), or in the presence of 10 mg/ml of an unrelated mAb, mAb.MGr2 or mAb.MGr611. Data are expressed as percentage of binding as compared to that obtained in the absence of any mAb. Average values (and the range) from two independent experiments are reported. (d) Inhibition of NL1.1-luc gene transduction by anti-HER2 mAb. NL1.1-luc phage (4×1010 TU) was incubated with HER2-positive SKBr3 cells for four hours in the absence (white bar) or in the presence of 10 mg/ml of an unrelated mAb, mAb.MGr2 or mAb.MGr6. Then unbound phage were removed, the cells were washed and incubated for 48 hours before luciferase assay. Data are expressed as percentage of luciferase activity obtained in the absence of any mAb. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported.</note><note type="content">Figure 7: Effect of incubation time on NL1.1-luc transfection efficiency. A total of 5×1010 TU of NL1.1-luc (filled circles) or wt-luc (empty circles) phage was incubated at 37°C with SKBr3 cells for increasing times. Then unbound phage were removed, the cells were washed and incubated for 48 hours before luciferase assay. Each point represents the mean of triplicate determinations and the standard deviation for each value is reported. Luciferase activity in mock-transfected controls was 2.4×104 (±1.7×103) RLU/mg protein.</note><note type="content">Table 1: Transfection efficiency in SKBr3 cells</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>phage display</topic><topic>targeting</topic><topic>HER2/neu receptor</topic><topic>gene delivery</topic><topic>cancer</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>AP : alkaline phosphatase</topic><topic>BSA : bovine serum albumin</topic><topic>CAR : Coxsackie-adenovirus receptor</topic><topic>CMV : human cytomegalovirus</topic><topic>dsDNA : double-stranded DNA</topic><topic>ECD : extracellular domain</topic><topic>FACS : fluorescence-activated cell sorting</topic><topic>FCS : fetal calf serum</topic><topic>HER2 : human epidermal growth factor receptor 2</topic><topic>HRP : horseradish peroxidase</topic><topic>mAb : monoclonal antibody</topic><topic>RLU : relative light units</topic><topic>ssDNA : single-stranded DNA</topic><topic>TU : transducing units</topic><topic>wt : wild-type</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0259-8</identifier><part><date>2001</date><detail type="volume"><number>313</number><caption>vol.</caption></detail><detail type="issue"><number>5</number><caption>no.</caption></detail><extent unit="issue-pages"><start>923</start><end>1206</end></extent><extent unit="pages"><start>965</start><end>976</end></extent></part></relatedItem><identifier type="istex">1E7ADEACE591D8C14BF36A74093423AAF93C7BC6</identifier><identifier type="ark">ark:/67375/6H6-T9090RX7-L</identifier><identifier type="DOI">10.1006/jmbi.2001.5111</identifier><identifier type="PII">S0022-2836(01)95111-3</identifier><accessCondition type="use and reproduction" contentType="copyright">©2001 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©2001</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-T9090RX7-L/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002084 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 002084 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Istex
|étape= Corpus
|type= RBID
|clé= ISTEX:1E7ADEACE591D8C14BF36A74093423AAF93C7BC6
|texte= Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage
}}
| This area was generated with Dilib version V0.6.33. |  |