3 NUCLEAR REPROGRAMMING OF PORCINE FIBROBLAST CELLS BY XENOPUS EGG EXTRACTS

Abstract

Much evidence indicates that somatic cells can be reprogrammed in an oocyte cytoplasm. The nuclear reprogramming consists of many unknown processes, and mechanisms underlying these processes still remain to be elucidated. Recently some reports noted that Xenopus oocytes or eggs can induce some of the reprogramming events in mammalian cells. We investigated the processes of nuclear reprogramming of porcine fibroblast cells by Xenopus egg extracts to understand how egg extracts trigger the reprogramming and/or dedifferentiation of cells. Unfertilized Xenopus eggs were collected from mature females. After removal of the jelly coat, activation was routinely achieved by calcium ionophore A23187. The eggs were immediately centrifuged and the cytoplasmic fraction was used as egg extracts. Porcine fibroblast cells were permeabilized by streptolysin O and incubated in the egg extracts under the ATP-generating system (1 mM ATP, 5 mM phosphocreatine, and 20 U/mL creatine kinase) for 30 min at 37°C or 2 h at 23°C. The incorporation of Xenopus-specific linker histone B4 into porcine fibroblasts was examined by immunofluorescence and immunobloting analysis. After collection of cells from the extracts, permeabilized membranes of the cells were resealed in culture medium containing 2 mM CaCl₂ for 2 h. The cells were then incubated in DMEM with 10% fetal bovine serum (FBS) or porcine zygote medium-3 (PZM-3: Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) containing 5.55 mM glucose and 5% FBS. RNAs were extracted from the cells in each culture dish and Oct-4 expression was examined by RT-PCR analysis every day until Day 8. The primers were designed to span the 99 base-pair intron region of porcine Oct-4 gene for recognizing both spliced and unspliced transcripts. The incorporation of histone B4 from Xenopus egg extracts was observed at the nuclear region of the porcine fibroblasts under both the 37°C and the 23°C conditions. Because the histone B4 incorporation was inhibited by addition of Apyrase, an ATPase, a part of reprogramming might be an ATP-dependent process. When treated cells were incubated in DMEM or PZM-3, Oct-4 expression was detected in the cells cultured in DMEM, but not in PZM-3. However, the transcripts of Oct-4 were mainly obtained in unspliced form at the earlier stage of culture (after Day 1 to Day 4 of culture), suggesting that a part of reprogramming processes by the egg extracts involves induction of dedifferention of cells or activation of a pluripotent marker gene such as Oct-4. Xenopus egg extract may provide a system to investigate the processes involving nuclear reprogramming and the pluripotent state of mammalian cells in vitro.