Production of Transgenic F0Animals and Permanent Lines by Sperm Nuclear Transplantation inXenopus tropicalis

Abstract

Early efforts in the 1980s showed that DNA microinjected intoXenopusembryos could be integrated into the genome and transmitted through the germline at low efficiency. Subsequent studies revealed that transgenic lines, typically with multiple-copy inserts (e.g., to develop bright fluorescent protein-reporter lines), could be created via sperm nuclear injection protocols such as the one entitled restriction enzyme-mediated insertion, or REMI. Here we describe a refined sperm nuclear injection procedure, with a number of alterations, including elimination of a potential DNA-damaging restriction enzyme treatment, aimed at making F0transgenic animals and transgenic lines inXenopus tropicalis. This protocol also uses an oocyte extract rather than the egg extract used in older protocols. These changes simplify and improve the efficiency of the procedure.