23 REPROGRAMMING MAMMALIAN CELLS IN XENOPUS EGG EXTRACTS: ROLE OF EMBRYONIC LAMIN B3

Abstract

The generation of animals by nuclear transplantation has demonstrated that a fully differentiated cell can be reversed into totipotency when transferred into an oocyte. Identification of oocyte specific molecules responsible for the reprogramming of somatic cells may contribute to the understanding of cell differentiation and embryo development. We have developed a heterologous system to investigate the effect of lamin B3, a major component of Xenopus laevis egg cytoplasm, on DNA replication of mammalian somatic cells. Bovine fetal fibroblasts were arrested at G1/S by incubation in aphidicolin for 18 h. After permeabilization with digitonin, the cells were incubated in either (1) lamin B3 depleted, or (2) whole Xenopus egg extracts (1000 cells μL⁻¹ extract) supplemented with an energy regenerating system for a period of 3 h at 21°C. Xenopus lamin B3-depleted egg extracts were prepared by three rounds of incubation with Dynabeads coated with a mouse monoclonal lamin B3 antibody (mAbLB3). Immunodepletion was confirmed by western blotting. Purified lamin B3 was obtained by dialysis of the beads after immunodepletion, and the purified lamin B3 was used for rescue experiments. DNA replication of cells incubated in the extracts was assessed by adding 25 μM Biotin-11-dUTP for 3 h. After treatment cells were fixed in 70% methanol at −20°C and incubated in mAbLB3 for 30 min at 37°C. This was followed by incubation in FITC-conjugated sheep anti-mouse antibody and in 5 mg mL⁻¹ Texas Red-conjugated Streptavidin for 40 min at 37°C. After three hours’ incubation in egg extracts, DNA replication was detected in 60% of cells and more than 95% of cells were lamin B3 positive. In contrast, DNA replication in immunodepleted extracts was significantly lower (P ≤ 0.01, by one-way ANOVA) than in cells incubated in whole extracts and was coincident with the few lamin B3-positive cells observed. More than 95% of cells were lamin B3-negative and did not replicate DNA. When purified lamin B3 was re-added to depleted extracts, DNA replication was detected in 60% of cells. DNA synthesis resumed in 93% of control cells 3 h after release from aphidicolin into culture medium at 39°C. These experiments show that somatic nuclei, which possess a nuclear envelope with somatic variants of lamins, are able to synthesize DNA in egg extracts only when Xenopus lamin B3 is incorporated into the nuclear envelope. This heterologous system provides new information on the role of an embryonic molecule, namely Xenopus lamin B3, in the reprogramming of DNA replication of somatic cells incubated in egg environment. These results open new questions as to whether embryonic lamins also exist in mammals, and whether failure in development of cloned animals is in part due to abnormal or incomplete replacement of somatic variants of proteins with their embryonic counterparts.