RESUMO
BACKGROUND: Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development. RESULTS: SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses. CONCLUSIONS: Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.
RESUMO
BACKGROUND: Autophagy regulation involves an intricate network that can degrade and recycle cytosolic components in autophagosomes when cells are subject to various stress signals. p53 plays a dual role of induction or inhibition in the regulation of autophagy. Recently, pigs have been considered an excellent large animal model for their many anatomical and physiological similarities to humans. Here, we investigated the relationship between p53 and autophagy, as well as the underling molecular basis, in porcine fibroblast cells (PFCs). METHODS: Autophagy was induced by Earle's balanced salt solution (EBSS) in p53-/- and p53wt PFCs. The autophagy response was assessed by immunoblotting, transmission electron microscopy (TEM) and fluorescent staining. The molecular basis for p53 regulation of autophagy was analyzed by qPCR. RESULTS: We found that the increased expression of LC3-II and the decreased expression of P62 occurred earlier in p53-/- PFCs than in p53wt PFCs, the relative autophagic flux of p53-/- PFCs was stronger than that of p53wt PFCs in a time-dependent manner. Meanwhile, we observed a visualized increase of autophagosomes in p53-/- PFCs. Moreover, we found greater accumulation of LC3 punctate and acidic vesicular organelle (AVOs) in the cytoplasm of p53-/- PFCs than in that of p53wt PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression of 6 autophagy signaling pathway-related genes and 14 autophagy-related (ATG) genes by qPCR. We found that the expression patterns of the 6 genes had significant differences in both groups of treated PFCs. These results demonstrated that p53 negatively regulated autophagy and involving the downregulation of LMNA gene by p53 via an unknown pathway, which causes the upregulation of the LC3, ULK1, ATG4B, ATG16L1 and ATG9A genes and the downregulation of the P62 gene. CONCLUSIONS: p53-/- PFCs responded to autophagy earlier than p53wt PFCs, which implied that p53 might inhibit autophagy. The expression patterns of autophagy signaling pathway-related genes and ATG genes were most different between p53-/- and p53wt PFCs. Our study will provide a new biological model and contribute to further study of the molecular basis for p53 and autophagy.