With easy access to core facilities or commercial providers of pronuclear injections, generating simple Thy1-XFP transgenic mice (where XFP stands for any fluorescent protein) is now a possibility even for small laboratories. The generation of new Thy1 transgenic lines generally consists of five steps: (1) engineering and characterization of the desired fluorescent reporter protein, (2) cloning of the reporter protein into the Thy1 vector, (3) linearization and purification of the new Thy1 construct, (4) pronuclear injection to generate founders, and (5) screening of founder progeny to establish transgenic lines. Here, we provide a protocol for Steps 2 and 3. The sequence for a desired fluorescent reporter protein is cloned into the XhoI restriction site of the Thy1 vector. This usually involves blunt-end cloning because the traditional Thy1 vector does not carry an intact multiple cloning site. Following successful cloning, the DNA is prepared for pronuclear injection by linearizing it using EcoRI and PvuI restriction enzymes. The purified linearized DNA must then be sent to a facility specializing in pronuclear injection to generate transgenic founder mice.