ABSTRACT
The aim of this study was to isolate Embryonic Stem Cells (ESCs) from native chicken and to characterize their pluripotency properties through the cellular and molecular markers. Samples obtained from fertilized eggs from Mazandaran native hens. Cells were isolated from area of pellucida from stage X native hens' blastoderm. Then the cells were cultured on inactivated mouse SNL feeder cells in the presence of LIF, IGF-1, bFGF, CNTF, OSM, SCF, Il-6, and Il-11 growth factors. The native chickens' ESCs colonies were picked up and subsequently passaged. To characterize the cells, they were analyzed for their alkaline phosphatase activity, and also for the expression of SSEA-4, and TRA-1-60 as embryonic-specific markers at the protein level. Furthermore, the expression of pluripotency (cPouV, Sox2, and Nanog) and cell lineage specific (Cvh, Brachyury, and Gata6) gene markers was evaluated at the level of mRNA using quantitative RT-PCR. Isolated cells were passaged repeatedly and successfully up to ten passages. The stemness of embryonic cells has been approved by the activity of the alkaline phosphatase, presence of the SSEA-4, and TRA-1-60 protein, and expression of the molecular marker (cPouV, Nanog, and Sox-2) genes. The spontaneous differentiation of chicken ESCs confirmed the pluripotecy of the cells in differentiation into specialized cell lineages. Our observation showed that ESCs can be isolated successfully from stage X blastoderm of Mazandaran native chickens and these cells maintain their stemness properties during multi-passages in vitro.
Subject(s)
Chickens , Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Alkaline Phosphatase/metabolism , Animals , Biomarkers/metabolism , Blastoderm , Cell Culture Techniques , Cell Differentiation , Cell Lineage , Coculture Techniques , Embryonic Stem Cells/metabolism , Intercellular Signaling Peptides and Proteins , Mice , Pluripotent Stem Cells/metabolism , Stage-Specific Embryonic Antigens/metabolismABSTRACT
DNA vaccination using a plasmid encoding Human rotavirus A (HuRV-A) inner capsid VP2 was examined in a mouse model. BALB/c mice were immunized intranasally (i.n.) with a VP2 DNA vaccine that induced cellular and humoral immune response to HuRV-A. The increased levels of cytokines IFN-gamma and IL-4 and the production of anti-VP2 IgG antibodies were detected. In addition, splenocyte proliferation detected by MTT test was enhanced in the mice treated with the vaccine. These results may encourage the development of a HuRV-A DNA vaccine derived from the inner layer of viral capsid that can be administered i.n.
Subject(s)
Capsid Proteins/immunology , Rotavirus Infections/immunology , Rotavirus/immunology , Viral Vaccines/immunology , Administration, Intranasal , Animals , Capsid Proteins/administration & dosage , Capsid Proteins/genetics , Female , Humans , Immunity, Humoral , Immunization , Male , Mice , Mice, Inbred BALB C , Rotavirus/genetics , Rotavirus Infections/virology , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/geneticsABSTRACT
A system for the expression and characterization of VP2 protein of Human rotavirus A, strain G3 was established in the mammalian lung cell line (A549). Expression of the recombinant VP2 (rVP2) protein was detected 48-72 hrs after transfection by Western blotting. The rVP2 protein expressed in A549 cells formed intracellular core-like particles (CLPs) 30 nm in diameter detected by electron microscopy (EM). These results showed that the A549 cells are suitable as efficient eukaryotic host for production of rVP2 protein.
