This protocol describes an example of complete zygote enucleation and transplantation of male and female pronuclei; however, single pronuclei can also be removed and transplanted. In this method, pronuclei are removed without penetrating the plasma membrane of the zygote. Instead, they are withdrawn individually or together into a membrane-bound karyoplast that can then be fused with a recipient enucleated zygote using inactivated Sendai virus or electrofusion. Preincubation of the embryos in the presence of the cytoskeletal inhibitors cytochalasin B and colcemid is critical for the survival of the embryos during this microsurgical procedure. The protocol is divided into five parts: (1) isolating embryos, (2) making an enucleation/injection pipette, (3) enucleating a zygote, (4) preparing inactivated Sendai virus, and (5) introducing pronuclei into enucleated zygotes.
© 2017 Cold Spring Harbor Laboratory Press.