Abstract
Rabbit embryonic stem (rES) cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES) cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES) cells, and parthenote-derived ES (p-rES) cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (p<0.05). Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%), nucleus (14%), and cytoskeletal machineries (13%). These proteins were majorly involved in biological functions of energy and metabolic pathways (25%), cell growth and maintenance (25%), signal transduction (14%), and protein metabolisms (10%). When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins), structural molecule (KRT8), catalytic molecules (α-enolase), receptor complex scaffold (14-3-3 protein sigma), microfilament motor proteins (Myosin-9), and heat shock protein (HSP60), were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (p<0.05). Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins differentially expressed between f-rES and p-rES cells. These analyses provide insights into rES cell biology and would invite more in-depth studies toward rES cell applications.
Highlights
Rabbit embryonic stem cells are pluripotent cells derived from the blastocyst stage embryos [1,2]
Among the 54 spots, the expression levels of 29 spots or proteins represent the proteins with expression levels that are higher in f-Rabbit embryonic stem (rES) and parthenote-derived rES (p-rES) cells than in fibroblasts (Table 1)
While the gene and protein expressions underlying the pluripotency of fertilized embryos (f-rES) and p-rES cells are largely unknown, this study investigated the protein profiles of these cell lines by a proteomics approach using rabbit fibroblast cells as the control
Summary
Rabbit embryonic stem (rES) cells are pluripotent cells derived from the blastocyst stage embryos [1,2]. Activated (PA) oocytes or embryos are subjected to artificial stimuli to initiate embryonic development without fertilization process or incorporation of sperm chromosomes These parthenotes possess chromosomes entirely of the maternal origin and fail to develop to term due to a lack of paternal gene expressions or normal genomic imprinting [10,11]. Similar to f-rES cells, parthenote-derived rES (p-rES) cells can continuously proliferate in vitro, retain self-renewal capacity without differentiation [3,4,12], and differentiate into cell lineages of the three germ layers both in vitro and in vivo They expressed the same set of pluripotency marker genes (Oct, Nanog, and Sox2), alkaline phosphatase (AP), and proteins such as cell surface markers including stage specific embryonic antigen-4
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