Generation Of Human Embryonic Stem Cell Lineexpressing GFP In Cholinergic Neurons Using CRISPR/cas9 System

Cholinergic neurons, exist widespreadly in organism, are able to synthesize acetycholine which is one of neurotransmitters. Cholinergic neurons in central nervous system are closely related to learning, memory and attention. Pathological lesion of cholinergic neurons cause Alzheimer’s disease(AD), a severe neurodegenerative diseases. Cholinergic neuron-labeled cell lines are versatile tools for biological studies on differentiation of cholinergic neurons, physiological and biochemical of cholinergic neurons and pathological mechanism of AD. Although cholinergic neuron-labeled cell lines has been well developed in rat and mouse, lack of feasible human cell lines is becoming a bottleneck for cholinergic neuron-relevant research.Here we report the generation of clonally derived ChAT-zsGreen reporter hESC line that express zs Green under the control of the Choline acetyltransferase(Ch AT) promoter using CRISPR(Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the Ch AT-zs Green h ESC reporter cell lines retain the features of undifferentiated h ESC. After neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein(GFP). The Ch AT-zs Green reporter h ESC lines are invaluable not only for the study of the cholinergic neurons but also contribute to the establish of other specific reporter cell lines.