Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.
Bibliographical noteFunding Information:
We thank Ammon Fager and Jin Liu for initial work in our lab on this method, and Darin Coombs for help with protocols pertaining to the care of Xenopus colonies. This work was supported by the National Institutes of Health (GM 61275 to K.S.U.), National Institute of Health training grant (5 T32 GM07464 to M.M.H.), and predoctoral fellowships from the National Science Foundation and the University of Utah (A.J.P).
- Nuclear envelope breakdown
- Nuclear membrane
- Nuclear pore
- Sperm chromatin DNA
- Xenopus egg extract