A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells
Identifieur interne : 001F38 ( Istex/Corpus ); précédent : 001F37; suivant : 001F39A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells
Auteurs : Fabrice Ango ; Serenella Albani-Torregrossa ; Cécile Joly ; David Robbe ; Jean-Marie Michel ; Jean-Philippe Pin ; Joël Bockaert ; Laurent FagniSource :
- Neuropharmacology [ 0028-3908 ] ; 1999.
English descriptors
- KwdEn :
- Teeft :
- Ango, Bockaert, Cdna, Cerebellar, Cerebellar cultures, Cerebellar granule cells, Cerebellar granule cells transfected, Channel activity, Cotransfection, Culture medium, Cultured cerebellar granule cells, Expression plasmid, Expression plasmids, Fagni, Functional expression, Gene transfer, Glial, Glial cells, Glutamate, Granule, Granule cells, Hippocampal neurons, Intracellular, Metabotropic, Metabotropic glutamate receptors, Mglu, Mglu receptor agonist, Mglu receptors, Mglu1, Mglu1 receptor, Mglu5, Mglu5 receptor, Mglu5 receptor protein, Native mglu1 receptor, Neuron, Neuronal, Neuronal cultures, Neurones, Neuropharmacology, Neurosci, Petri, Petri dish, Plasmid, Plating, Primary culture, Primary cultures, Reagent, Receptor, Room temperature, Similar results, Table value, Transfast, Transfast ratio, Transfected, Transfected cells, Transfected granule cells, Transfection, Transfection method, Transfection rate, Transfection reagents, Uorescent, Uorescent cells, Uorescent protein.
Abstract
Abstract: We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.
Url:
DOI: 10.1016/S0028-3908(99)00005-2
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ISTEX:F1D1DDCE9EFCC7D09B3D9DA81201DD748953473ALe document en format XML
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Pharmacology, University of Florence, Viale Morgagni, 65, 50134 Florence, Italy</mods:affiliation></affiliation></author><author><name sortKey="Joly, Cecile" sort="Joly, Cecile" uniqKey="Joly C" first="Cécile" last="Joly">Cécile Joly</name><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Robbe, David" sort="Robbe, David" uniqKey="Robbe D" first="David" last="Robbe">David Robbe</name><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Michel, Jean Marie" sort="Michel, Jean Marie" uniqKey="Michel J" first="Jean-Marie" last="Michel">Jean-Marie Michel</name><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Pin, Jean Philippe" sort="Pin, Jean Philippe" uniqKey="Pin J" first="Jean-Philippe" last="Pin">Jean-Philippe Pin</name><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Bockaert, Joel" sort="Bockaert, Joel" uniqKey="Bockaert J" first="Joël" last="Bockaert">Joël Bockaert</name><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Fagni, Laurent" sort="Fagni, Laurent" uniqKey="Fagni L" first="Laurent" last="Fagni">Laurent Fagni</name><affiliation><mods:affiliation>E-mail: fagni@bacchus.montp.inserm.fr</mods:affiliation></affiliation><affiliation><mods:affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Neuropharmacology</title><title level="j" type="abbrev">NP</title><idno type="ISSN">0028-3908</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">38</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="793">793</biblScope><biblScope unit="page" to="803">803</biblScope></imprint><idno type="ISSN">0028-3908</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0028-3908</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Big K+ channels</term><term>Culture</term><term>Green fluorescent protein</term><term>Metabotropic glutamate receptor</term><term>Neurones</term><term>Transfection</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Ango</term><term>Bockaert</term><term>Cdna</term><term>Cerebellar</term><term>Cerebellar cultures</term><term>Cerebellar granule cells</term><term>Cerebellar granule cells transfected</term><term>Channel activity</term><term>Cotransfection</term><term>Culture medium</term><term>Cultured cerebellar granule cells</term><term>Expression plasmid</term><term>Expression plasmids</term><term>Fagni</term><term>Functional expression</term><term>Gene transfer</term><term>Glial</term><term>Glial cells</term><term>Glutamate</term><term>Granule</term><term>Granule cells</term><term>Hippocampal neurons</term><term>Intracellular</term><term>Metabotropic</term><term>Metabotropic glutamate receptors</term><term>Mglu</term><term>Mglu receptor agonist</term><term>Mglu receptors</term><term>Mglu1</term><term>Mglu1 receptor</term><term>Mglu5</term><term>Mglu5 receptor</term><term>Mglu5 receptor protein</term><term>Native mglu1 receptor</term><term>Neuron</term><term>Neuronal</term><term>Neuronal cultures</term><term>Neurones</term><term>Neuropharmacology</term><term>Neurosci</term><term>Petri</term><term>Petri dish</term><term>Plasmid</term><term>Plating</term><term>Primary culture</term><term>Primary cultures</term><term>Reagent</term><term>Receptor</term><term>Room temperature</term><term>Similar results</term><term>Table value</term><term>Transfast</term><term>Transfast ratio</term><term>Transfected</term><term>Transfected cells</term><term>Transfected granule cells</term><term>Transfection</term><term>Transfection method</term><term>Transfection rate</term><term>Transfection reagents</term><term>Uorescent</term><term>Uorescent cells</term><term>Uorescent protein</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>receptor</json:string><json:string>transfection</json:string><json:string>granule</json:string><json:string>cerebellar</json:string><json:string>transfected</json:string><json:string>cdna</json:string><json:string>mglu5</json:string><json:string>transfast</json:string><json:string>neurones</json:string><json:string>plasmid</json:string><json:string>mglu5 receptor</json:string><json:string>cerebellar granule cells</json:string><json:string>uorescent</json:string><json:string>mglu1</json:string><json:string>granule 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neurons</json:string><json:string>gene transfer</json:string><json:string>mglu receptors</json:string></teeft></keywords><author><json:item><name>Fabrice Ango</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>Serenella Albani-Torregrossa</name><affiliations><json:string>Department of Pharmacology, University of Florence, Viale Morgagni, 65, 50134 Florence, Italy</json:string></affiliations></json:item><json:item><name>Cécile Joly</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>David Robbe</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>Jean-Marie Michel</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>Jean-Philippe Pin</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>Joël Bockaert</name><affiliations><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item><json:item><name>Laurent Fagni</name><affiliations><json:string>E-mail: fagni@bacchus.montp.inserm.fr</json:string><json:string>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Transfection</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Culture</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Neurones</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Metabotropic glutamate receptor</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Green fluorescent protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Big K+ channels</value></json:item></subject><arkIstex>ark:/67375/6H6-QLX111ZR-Z</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.</abstract><qualityIndicators><score>9.496</score><pdfWordCount>5578</pdfWordCount><pdfCharCount>35001</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>552 x 770 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>208</abstractWordCount><abstractCharCount>1410</abstractCharCount><keywordCount>6</keywordCount></qualityIndicators><title>A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells</title><pmid><json:string>10465683</json:string></pmid><pii><json:string>S0028-3908(99)00005-2</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Neuropharmacology</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0028-3908</json:string></issn><pii><json:string>S0028-3908(00)X0046-9</json:string></pii><volume>38</volume><issue>6</issue><pages><first>793</first><last>803</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1999</json:string></date><geogName></geogName><orgName><json:string>Axon Instruments</json:string><json:string>Elsevier Science Ltd.</json:string><json:string>Clontech</json:string><json:string>Italy Accepted</json:string><json:string>Bayer France/ Troponwerke Germany</json:string><json:string>CNRS</json:string><json:string>CEE BIOMED II</json:string><json:string>BIOTECH II</json:string></orgName><orgName_funder><json:string>CNRS</json:string><json:string>CEE BIOMED II</json:string><json:string>BIOTECH II</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>L. Fagni</json:string><json:string>S.E.M. Given</json:string><json:string>Laurent Charvet</json:string><json:string>B. Mouillac</json:string><json:string>Bernard Mouillac</json:string><json:string>Jean-Philippe Pin</json:string><json:string>B. Note</json:string></persName><placeName><json:string>Foster City</json:string><json:string>UK</json:string><json:string>Mannheim</json:string><json:string>USA</json:string><json:string>Strasbourg</json:string><json:string>CA</json:string><json:string>France</json:string><json:string>Hamamatsu</json:string><json:string>Belgium</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Boeckman et al., 1996</json:string><json:string>Kaech et al., 1996</json:string><json:string>Chavis et al. (1998)</json:string><json:string>Levi et al., 1984</json:string><json:string>Chavis et al., 1998</json:string><json:string>B. F. Ango et al.</json:string><json:string>Marshall et al., 1995</json:string><json:string>Horton et al., 1989</json:string><json:string>Pin and Duvoisin, 1995</json:string><json:string>Loeffler and Behr, 1993</json:string><json:string>Van-Vliet et al., 1989</json:string><json:string>Olkkonen et al., 1993</json:string><json:string>Fagni et al., 1991</json:string><json:string>Werner et al., 1990</json:string><json:string>F. Ango et al.</json:string><json:string>Prasher et al., 1992</json:string><json:string>Wilkemeyer et al., 1996</json:string><json:string>Joly et al.</json:string><json:string>Chavis et al., 1995</json:string><json:string>Fagni et al., 1995</json:string><json:string>Xia et al., 1996</json:string><json:string>Fagni et al. (1991)</json:string><json:string>Lambert et al., 1996</json:string><json:string>Weiss et al., 1986</json:string><json:string>Casabona et al., 1997</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-QLX111ZR-Z</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - pharmacology & pharmacy</json:string><json:string>2 - neurosciences</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - clinical medicine</json:string><json:string>3 - neurology & neurosurgery</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Neuroscience</json:string><json:string>3 - Cellular and Molecular Neuroscience</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Pharmacology</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></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1016/S0028-3908(99)00005-2</json:string></doi><id>F1D1DDCE9EFCC7D09B3D9DA81201DD748953473A</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-QLX111ZR-Z/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-QLX111ZR-Z/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-QLX111ZR-Z/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1999 Elsevier Science Ltd</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1999</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 type="content">Fig. 1: Morphological differences between cerebellar granule (neurone) and non-granule (glial) cells. Cells were transfected with pEGFP-N1 by using a Transfast/cDNA ratio of 2 μl/1 μg. The arrow indicates a non granule cell (glial cell) easily distinguishable from the round shaped granule cell bodies located next to the glial cell. The calibration bar represents 50 μm.</note><note type="content">Fig. 2: pEGFP-N1-transfected cerebellar cells. A and B: Same field of a cerebellar culture illuminated with normal (A) and epifluorescent (B) light. Arrows in A indicate the fluorescent cells of panel B and vice versa. C: Typical view of a larger field of cerebellar cells transfected with pEGFP-N1/Transfast (1 μg/3 μl).</note><note type="content">Fig. 3: Transfast-mediated transfer of pEGFP-N1 cDNA into different primary cultures. Typical fluorescent neurones obtained from primary cultures of colliculus (A), striatum (B) and cerebellum (C and D). C and D: Typical Purkinje-like (C) and granule (D) cells. Purkinje-like cells were only occasionally found in our cerebellar cultures. All cultures were transfected by using cDNA/Transfast at a ratio of 1 μg/3 μl.</note><note type="content">Fig. 4: Voltage-activated K+, Ca2+ and Na+ membrane currents were not altered in pEGFP-N1-transfected cerebellar granule cells. Same legend as in Table 5. Each trace is an average of ten consecutive events. For each type of current, note the similarities in the kinetics between transfected and non transfected cells. For each line, the scale bar in the first column applies for the two following columns. Neurones were transfected using a cDNA/Transfast ratio of 1 μg/3 μl.</note><note type="content">Fig. 5: Cotransfection of cerebellar granule cells with pEGFP-N1 and pRKG5a-NHA cDNAs. Typical view of a cerebellar culture transfected with pEGFP-N1/pRKG5a-NHA cDNAs (1/2). Panel A shows a cerebellar granule cell expressing GFP. Panel B (same field as in A) shows granule cells expressing HA-tagged mGlu5 receptor, labelled with fluorescent Texas red secondary antibody. The arrow in panel B indicates the fluorescent cell of panel A. Panel C shows superimposed fluorescence of panels A and B. Note the larger number of cells expressing the mGlu5 receptor (red) compare to GFP (green).</note><note type="content">Fig. 6: Functional expression test of transfected pRKG5a-NHA in cultured cerebellar granule cell. A: The neurone was transfected with pEGFP-N1 using Transfast. Traces represent single big K+ channel activity recorded under the cell-attached configuration of the patch-clamp technique. Horizontal bars represent drug applications. Note that the agonistic effect of the group I mGlu receptor agonist, DHPG (400 μM) was blocked by the mGlu1 receptor antagonist CPCCOEt (250 μM). Similar results were obtained in five out of five cells. B: Same legend as in A except that this neurone was cotransfected with pEGFP-N1 and pRKG5a-NHA. Note the agonistic effect of DHPG in the presence of CPCCOEt. Similar results were found in five out of seven cells. For both cells, the channel was recorded at a holding pipette potential of −80 mV (putative membrane potential of +20 mV). Scale bars apply for both panels, A and B.</note><note type="content">Table 1: Effect of increasing the amount of Transfast on transfection efficiency in cerebellar culturea</note><note type="content">Table 2: Effect of increasing amount of pEGFP-N1 cDNA on transfection efficiencya</note><note type="content">Table 3: Delaying the day of transfection after plating decreased efficiency of Transfast in cerebellar granule cellsa</note><note type="content">Table 4: Comparison between different reagents on the transfer efficiency of pEGFP-N1 cDNA into cerebellar granule cellsa</note><note type="content">Table 5: Transfer of pEGFP-N1 cDNA into cerebellar granule cells by using Transfast did not alter properties of voltage-activated currentsa</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells</title><author xml:id="author-0000"><persName><forename type="first">Fabrice</forename><surname>Ango</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Serenella</forename><surname>Albani-Torregrossa</surname></persName><affiliation>Department of Pharmacology, University of Florence, Viale Morgagni, 65, 50134 Florence, Italy</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Cécile</forename><surname>Joly</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0003"><persName><forename type="first">David</forename><surname>Robbe</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Jean-Marie</forename><surname>Michel</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Jean-Philippe</forename><surname>Pin</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Joël</forename><surname>Bockaert</surname></persName><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Laurent</forename><surname>Fagni</surname></persName><email>fagni@bacchus.montp.inserm.fr</email><note type="correspondence"><p>Corresponding author. Tel.: +33-467-142945; fax: +33-467-542432</p></note><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation></author><idno type="istex">F1D1DDCE9EFCC7D09B3D9DA81201DD748953473A</idno><idno type="ark">ark:/67375/6H6-QLX111ZR-Z</idno><idno type="DOI">10.1016/S0028-3908(99)00005-2</idno><idno type="PII">S0028-3908(99)00005-2</idno></analytic><monogr><title level="j">Neuropharmacology</title><title level="j" type="abbrev">NP</title><idno type="pISSN">0028-3908</idno><idno type="PII">S0028-3908(00)X0046-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">38</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="793">793</biblScope><biblScope unit="page" to="803">803</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Transfection</term></item><item><term>Culture</term></item><item><term>Neurones</term></item><item><term>Metabotropic glutamate receptor</term></item><item><term>Green fluorescent protein</term></item><item><term>Big K+ channels</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999">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-QLX111ZR-Z/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="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>NP</jid><aid>1059</aid><ce:pii>S0028-3908(99)00005-2</ce:pii><ce:doi>10.1016/S0028-3908(99)00005-2</ce:doi><ce:copyright type="full-transfer" year="1999">Elsevier Science Ltd</ce:copyright></item-info><head><ce:title>A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu<ce:inf>5</ce:inf> receptor in cerebellar granule cells</ce:title><ce:author-group><ce:author><ce:given-name>Fabrice</ce:given-name><ce:surname>Ango</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Serenella</ce:given-name><ce:surname>Albani-Torregrossa</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Cécile</ce:given-name><ce:surname>Joly</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>David</ce:given-name><ce:surname>Robbe</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Jean-Marie</ce:given-name><ce:surname>Michel</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Jean-Philippe</ce:given-name><ce:surname>Pin</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Joël</ce:given-name><ce:surname>Bockaert</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Laurent</ce:given-name><ce:surname>Fagni</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>fagni@bacchus.montp.inserm.fr</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Department of Pharmacology, University of Florence, Viale Morgagni, 65, 50134 Florence, Italy</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +33-467-142945; fax: +33-467-542432</ce:text></ce:correspondence></ce:author-group><ce:date-accepted day="23" month="12" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K<ce:sup>+</ce:sup> channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu<ce:inf>5</ce:inf> receptor). Ninety percent of the cells expressing GFP also expressed mGlu<ce:inf>5</ce:inf> receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu<ce:inf>5</ce:inf> receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu<ce:inf>5</ce:inf> receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu<ce:inf>5</ce:inf> receptor induced single big K<ce:sup>+</ce:sup> channel activity, as it was the case for the native mGlu<ce:inf>1</ce:inf> receptor. This indicated that the transfected mGlu<ce:inf>5</ce:inf> receptor plasmid was functionally expressed and that both mGlu<ce:inf>1</ce:inf> and mGlu<ce:inf>5</ce:inf> receptors may share common coupling mechanisms to big K<ce:sup>+</ce:sup> channels in neurones.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Transfection</ce:text></ce:keyword><ce:keyword><ce:text>Culture</ce:text></ce:keyword><ce:keyword><ce:text>Neurones</ce:text></ce:keyword><ce:keyword><ce:text>Metabotropic glutamate receptor</ce:text></ce:keyword><ce:keyword><ce:text>Green fluorescent protein</ce:text></ce:keyword><ce:keyword><ce:text>Big K<ce:sup>+</ce:sup> channels</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu</title></titleInfo><name type="personal"><namePart type="given">Fabrice</namePart><namePart type="family">Ango</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Serenella</namePart><namePart type="family">Albani-Torregrossa</namePart><affiliation>Department of Pharmacology, University of Florence, Viale Morgagni, 65, 50134 Florence, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Cécile</namePart><namePart type="family">Joly</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">David</namePart><namePart type="family">Robbe</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jean-Marie</namePart><namePart type="family">Michel</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jean-Philippe</namePart><namePart type="family">Pin</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Joël</namePart><namePart type="family">Bockaert</namePart><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Laurent</namePart><namePart type="family">Fagni</namePart><affiliation>E-mail: fagni@bacchus.montp.inserm.fr</affiliation><affiliation>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France</affiliation><description>Corresponding author. Tel.: +33-467-142945; fax: +33-467-542432</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">1999</dateIssued><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.</abstract><note type="content">Fig. 1: Morphological differences between cerebellar granule (neurone) and non-granule (glial) cells. Cells were transfected with pEGFP-N1 by using a Transfast/cDNA ratio of 2 μl/1 μg. The arrow indicates a non granule cell (glial cell) easily distinguishable from the round shaped granule cell bodies located next to the glial cell. The calibration bar represents 50 μm.</note><note type="content">Fig. 2: pEGFP-N1-transfected cerebellar cells. A and B: Same field of a cerebellar culture illuminated with normal (A) and epifluorescent (B) light. Arrows in A indicate the fluorescent cells of panel B and vice versa. C: Typical view of a larger field of cerebellar cells transfected with pEGFP-N1/Transfast (1 μg/3 μl).</note><note type="content">Fig. 3: Transfast-mediated transfer of pEGFP-N1 cDNA into different primary cultures. Typical fluorescent neurones obtained from primary cultures of colliculus (A), striatum (B) and cerebellum (C and D). C and D: Typical Purkinje-like (C) and granule (D) cells. Purkinje-like cells were only occasionally found in our cerebellar cultures. All cultures were transfected by using cDNA/Transfast at a ratio of 1 μg/3 μl.</note><note type="content">Fig. 4: Voltage-activated K+, Ca2+ and Na+ membrane currents were not altered in pEGFP-N1-transfected cerebellar granule cells. Same legend as in Table 5. Each trace is an average of ten consecutive events. For each type of current, note the similarities in the kinetics between transfected and non transfected cells. For each line, the scale bar in the first column applies for the two following columns. Neurones were transfected using a cDNA/Transfast ratio of 1 μg/3 μl.</note><note type="content">Fig. 5: Cotransfection of cerebellar granule cells with pEGFP-N1 and pRKG5a-NHA cDNAs. Typical view of a cerebellar culture transfected with pEGFP-N1/pRKG5a-NHA cDNAs (1/2). Panel A shows a cerebellar granule cell expressing GFP. Panel B (same field as in A) shows granule cells expressing HA-tagged mGlu5 receptor, labelled with fluorescent Texas red secondary antibody. The arrow in panel B indicates the fluorescent cell of panel A. Panel C shows superimposed fluorescence of panels A and B. Note the larger number of cells expressing the mGlu5 receptor (red) compare to GFP (green).</note><note type="content">Fig. 6: Functional expression test of transfected pRKG5a-NHA in cultured cerebellar granule cell. A: The neurone was transfected with pEGFP-N1 using Transfast. Traces represent single big K+ channel activity recorded under the cell-attached configuration of the patch-clamp technique. Horizontal bars represent drug applications. Note that the agonistic effect of the group I mGlu receptor agonist, DHPG (400 μM) was blocked by the mGlu1 receptor antagonist CPCCOEt (250 μM). Similar results were obtained in five out of five cells. B: Same legend as in A except that this neurone was cotransfected with pEGFP-N1 and pRKG5a-NHA. Note the agonistic effect of DHPG in the presence of CPCCOEt. Similar results were found in five out of seven cells. For both cells, the channel was recorded at a holding pipette potential of −80 mV (putative membrane potential of +20 mV). Scale bars apply for both panels, A and B.</note><note type="content">Table 1: Effect of increasing the amount of Transfast on transfection efficiency in cerebellar culturea</note><note type="content">Table 2: Effect of increasing amount of pEGFP-N1 cDNA on transfection efficiencya</note><note type="content">Table 3: Delaying the day of transfection after plating decreased efficiency of Transfast in cerebellar granule cellsa</note><note type="content">Table 4: Comparison between different reagents on the transfer efficiency of pEGFP-N1 cDNA into cerebellar granule cellsa</note><note type="content">Table 5: Transfer of pEGFP-N1 cDNA into cerebellar granule cells by using Transfast did not alter properties of voltage-activated currentsa</note><subject lang="en"><genre>Keywords</genre><topic>Transfection</topic><topic>Culture</topic><topic>Neurones</topic><topic>Metabotropic glutamate receptor</topic><topic>Green fluorescent protein</topic><topic>Big K+ channels</topic></subject><relatedItem type="host"><titleInfo><title>Neuropharmacology</title></titleInfo><titleInfo type="abbreviated"><title>NP</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">1999</dateIssued></originInfo><identifier type="ISSN">0028-3908</identifier><identifier type="PII">S0028-3908(00)X0046-9</identifier><part><date>1999</date><detail type="volume"><number>38</number><caption>vol.</caption></detail><detail type="issue"><number>6</number><caption>no.</caption></detail><extent unit="issue-pages"><start>735</start><end>916</end></extent><extent unit="pages"><start>793</start><end>803</end></extent></part></relatedItem><identifier type="istex">F1D1DDCE9EFCC7D09B3D9DA81201DD748953473A</identifier><identifier type="ark">ark:/67375/6H6-QLX111ZR-Z</identifier><identifier type="DOI">10.1016/S0028-3908(99)00005-2</identifier><identifier type="PII">S0028-3908(99)00005-2</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Elsevier Science Ltd</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>Elsevier Science Ltd, ©1999</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-QLX111ZR-Z/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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|étape= Corpus
|type= RBID
|clé= ISTEX:F1D1DDCE9EFCC7D09B3D9DA81201DD748953473A
|texte= A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells
}}
| This area was generated with Dilib version V0.6.33. |  |