Protocol for blastomere injection to generate bilateral hemimosaic Xenopus tadpoles

Here, we present a protocol to generate bilateral hemimosaic Xenopus tadpoles that allows systematic, independent manipulation of pre- and postsynaptic neurons in the tadpole retinotectal projection. We describe the steps to perform in vitro fertilization, followed by blastomere injections for two different manipulations. Specifically, we detail procedures for exogenous gene expression (GCaMP6s) by mRNA microinjection and endogenous gene knockdown (GluN1) with antisense morpholino microinjection. For complete details on the use and execution of this protocol, please refer to Kesner et al.¹ and Li et al.².