Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

The CaMKΙΙα-Cre mouse lines, possibly the most used Cre lines in neuroscience, have resulted in over 800 articles to date. Here, we demonstrate that the second most widely used CaMKΙΙα-Cre line, Tg(Camk2a-cre)2Gsc (or CamiCre), shows ectopic overexpression of synaptotagmin 2, the most efficient Ca²⁺ sensor for fast synchronous neurotransmitter release, in excitatory presynapses of Cre⁺ brains. Moreover, the upregulation of immediate-early genes and genes incorporated in bacterial artificial chromosome (BAC) transgenes, such as L-proline transporter Slc6a7, was found in Cre⁺ hippocampus. The copy number and integration site of the transgene are suggested to have caused the aberrant gene expression in Cre⁺ brains. Most importantly, CamiCre⁺ mice showed functional phenotypes, such as hyperactivity and enhanced associative learning, suggesting that neural activities are affected. These unexpected results suggest difficulties in interpreting results from studies using the CamiCre line and raise a warning of potential pitfalls in using Cre driver lines in general.