# Protocol for blastomere injection to generate bilateral hemimosaic Xenopus tadpoles

## TL;DR

This paper provides a detailed protocol for creating Xenopus tadpoles with specific genetic manipulations in their neurons.

## Contribution

A new protocol for generating bilateral hemimosaic Xenopus tadpoles with targeted gene expression and knockdown.

## Key findings

- The protocol enables independent manipulation of pre- and postsynaptic neurons in the retinotectal projection.
- It includes methods for mRNA microinjection of GCaMP6s and morpholino injection for GluN1 knockdown.

## Abstract

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

•Steps for preparation of mRNA and MO solutions for blastomere injection•Procedures for collection and in vitro fertilization of Xenopus eggs•Instructions for blastomere injection of mRNA or MO•Guidance on screening hemimosaic animals resulting from blastomere injections

Steps for preparation of mRNA and MO solutions for blastomere injection

Procedures for collection and in vitro fertilization of Xenopus eggs

Instructions for blastomere injection of mRNA or MO

Guidance on screening hemimosaic animals resulting from blastomere injections

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.