Production and functional expression of an epitope-tagged human choline acetyltransferase.
cDNA containing the entire coding region of the human choline acetyltransferase gene (hChAT) was fused to the influenza virus hemagglutinin (HA) epitope preceded by a Kozak sequence. The recombinant HA-hChAT was then inserted into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter. After transient transfection into COS-1 cells, expression was assayed by Northern and Western blot analysis and immunofluorescence. The chimeric HA-hChAT protein was compared to native hChAT for its ability to synthesize acetylcholine. It behaves identically to unmodified hChAT showing that the HA epitope does not affect ChAT activity. This approach enables one to distinguish the expression of the HA-hChAT from endogenous ChAT. Genetically engineered cells that express a high level of HA-hChAT could be used as a promising experimental tool for gene transfer and neurografting techniques as well as to produce and study transgenic mice.
Robert
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Laboratoire de Génétique Moléculaire des Eucaryotes, CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique, INSERM, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.
Quirin-Stricker
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D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002795:Choline O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000939:Epitopes; D005819:Genetic Markers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine