Nuclear Assembly in Giant Unilamellar Vesicles Encapsulating Xenopus Egg Extract

Abstract 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.