Modification of choline acetyltransferase by integration of green fluorescent protein does not affect enzyme activity and subcellular distribution.
Choline acetyltransferase (ChAT) is widely used as a marker enzyme to identify cholinergic neurons in the central and peripheral nervous system and to study developmental changes. In order to visualize expression of ChAT directly we have generated a ChAT-green fluorescent protein (GFP) fusion construct. Upon transfection of COS-1 cells and cultured rat hippocampal neurons, transgenic enzymatically active ChAT-GFP is expressed and shows intrinsic fluorescence. In COS-1 cells the ChAT-GFP construct revealed a subcellular distribution indistinguishable from wild-type ChAT. In primary neurons the fluorescence was present in the soma and neuritic processes. Hence, this construct will be useful for analyzing the expression and subcellular distribution of ChAT-GFP in cell and tissue culture.
Rathenberg
Jan
J
Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany.
Gärtner
Annette
A
Koenen
Michael
M
Witzemann
Veit
V
D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D002799:Cholinergic Fibers / Q000648:ultrastructure; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism; D017403:In Situ Hybridization / Q000379:methods; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002795:Choline O-Acetyltransferase