A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells.
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.
Ango
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F
UPR CNRS 9023, CCIPE, Montpellier, France.
Albani-Torregrossa
S
S
Joly
C
C
Robbe
D
D
Michel
J M
JM
Pin
J P
JP
Bockaert
J
J
Fagni
L
L
D000818:Animals; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D004247:DNA / Q000235:genetics*; D018014:Gene Transfer Techniques; D051379:Mice; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics*; D015221:Potassium Channels / Q000502:physiology; D018094:Receptors, Metabotropic Glutamate / Q000502:physiology*
D015220:Calcium Channels; D015221:Potassium Channels; D018094:Receptors, Metabotropic Glutamate; D004247:DNA