Nuclear Assembly in Giant Unilamellar Vesicles Encapsulating Xenopus Egg Extract

The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, a method for assembling a nucleus in giant unilamellar vesicles (GUVs) is proposed. To induce reconstitution of the nucleus, the interphase egg extract of African clawed frogs Xenopus laevis is utilized, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. The GUV formation efficiency is enhanced by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs. Characterization of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. The approach offers a versatile platform for constructing artificial cellular systems that closely mimic eukaryotic cells.