Cnpy3 mice reveal neuronal expression of Cnpy3 in the brain.

Background: Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear.

New Method: We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain.

Results: 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy3 knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain.

Comparison With Existing Methods: Conventional Cnpy3 antibodies could not allow us to investigate the distribution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells.

Conclusions: Taken together, we demonstrated that Cnpy3 knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders.