A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells
Identifieur interne : 000269 ( France/Analysis ); précédent : 000268; suivant : 000270A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells
Auteurs : Fabrice Ango [France] ; Serenella Albani-Torregrossa [Italie] ; Cécile Joly [France] ; David Robbe [France] ; Jean-Marie Michel [France] ; Jean-Philippe Pin [France] ; Joël Bockaert [France] ; Laurent Fagni [France]Source :
- Neuropharmacology [ 0028-3908 ] ; 1999.
Descripteurs français
- Wicri :
- topic : Culture.
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
Affiliations:
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(région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></affiliation></author><author><name sortKey="Robbe, David" sort="Robbe, David" uniqKey="Robbe D" first="David" last="Robbe">David Robbe</name><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier</wicri:regionArea><placeName><region type="region" nuts="2">Occitanie (région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></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 wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier</wicri:regionArea><placeName><region type="region" nuts="2">Occitanie (région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></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 wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier</wicri:regionArea><placeName><region type="region" nuts="2">Occitanie (région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></affiliation></author><author><name sortKey="Bockaert, Joel" sort="Bockaert, Joel" uniqKey="Bockaert J" first="Joël" last="Bockaert">Joël Bockaert</name><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier</wicri:regionArea><placeName><region type="region" nuts="2">Occitanie (région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></affiliation></author><author><name sortKey="Fagni, Laurent" sort="Fagni, Laurent" uniqKey="Fagni L" first="Laurent" last="Fagni">Laurent Fagni</name><affiliation wicri:level="1"><country wicri:rule="url">France</country></affiliation><affiliation wicri:level="3"><country xml:lang="fr">France</country><wicri:regionArea>UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier</wicri:regionArea><placeName><region type="region" nuts="2">Occitanie (région administrative)</region><region type="old region" nuts="2">Languedoc-Roussillon</region><settlement type="city">Montpellier</settlement></placeName></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><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Culture</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><affiliations><list><country><li>France</li><li>Italie</li></country><region><li>Languedoc-Roussillon</li><li>Occitanie (région administrative)</li></region><settlement><li>Montpellier</li></settlement></list><tree><country name="France"><region name="Occitanie (région administrative)"><name sortKey="Ango, Fabrice" sort="Ango, Fabrice" uniqKey="Ango F" first="Fabrice" last="Ango">Fabrice Ango</name></region><name sortKey="Bockaert, Joel" sort="Bockaert, Joel" uniqKey="Bockaert J" first="Joël" last="Bockaert">Joël Bockaert</name><name sortKey="Fagni, Laurent" sort="Fagni, Laurent" uniqKey="Fagni L" first="Laurent" last="Fagni">Laurent Fagni</name><name sortKey="Fagni, Laurent" sort="Fagni, Laurent" uniqKey="Fagni L" first="Laurent" last="Fagni">Laurent Fagni</name><name sortKey="Joly, Cecile" sort="Joly, Cecile" uniqKey="Joly C" first="Cécile" last="Joly">Cécile Joly</name><name sortKey="Michel, Jean Marie" sort="Michel, Jean Marie" uniqKey="Michel J" first="Jean-Marie" last="Michel">Jean-Marie Michel</name><name sortKey="Pin, Jean Philippe" sort="Pin, Jean Philippe" uniqKey="Pin J" first="Jean-Philippe" last="Pin">Jean-Philippe Pin</name><name sortKey="Robbe, David" sort="Robbe, David" uniqKey="Robbe D" first="David" last="Robbe">David Robbe</name></country><country name="Italie"><noRegion><name sortKey="Albani Torregrossa, Serenella" sort="Albani Torregrossa, Serenella" uniqKey="Albani Torregrossa S" first="Serenella" last="Albani-Torregrossa">Serenella Albani-Torregrossa</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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