Gene targeting and single-cell electroporation to analyze cholinergic neurons using choline acetyltransferase as marker

Cholinergic neurons use the neurotransmitter acetylcholine (ACh) and are involved in basic brain functions like learning and memory formation, awareness, the sleep and awaking cycle as well as motor behavior. Cholinergic neuron loss occurs during severe neurodegenerative diseases like Alzheimer or amyotrophic lateral sklerosis. So far immunohistochemistry or in situ hybridization against the cholinergic marker enzyme choline acetyltransferase (ChAT) serve to identify cholinergic neurons. However, these methods require fixation of the tissue and thus preclude in vivo experiments. One aim of the present work was to produce a fusion protein between ChAT and the green fluorescent protein (GFP) which will enable to visualize the cholinergic marker without fixation. Therefore the cDNA of ChAT was isolated from mouse spinal cord and subsequently the cDNA of GFP was cloned into a unique HindIII restriction site corresponding to the N-terminal part of the ChAT protein. The ChAT-GFP protein was expressed in COS-1 cells and primary cultures of hippocampal neurons as well as in hippocampal neurons of organotypic brain slice cultures. It was shown that the enzyme activity of the recombinant ChAT-GFP fusion protein is slightly reduced compared to the wildtype enzyme. However, it is still functional and similar distributed as the wildtype protein in cultured cells. In hippocampal neurons ChAT-GFP translocates into axonal processes and it is present in the presynaptic compartments. Since it was shown that ChAT-GFP is functional like the wildtype enzyme the cDNA of GFP was targeted to the ChAT gene of mouse embryonic stem cells by homologous recombination. These ChAT(GFP/+) ES cells were injected into blastozysts to produce chimeric mice. This blastozyst transfer provided 14 chimeric animals. Hetero- and homozygous mice which emerge from ChAT(GFP/+) ES cells will express the ChAT-GFP fusion protein in all cholinergic cells and hence these cholinergic neurons should show endogenous fluorescence. In parallel another ES cell clone was produced in which the first coding exon of ChAT is flanked by loxP sites (genotype ChAT(loxP/+)). Thi