Embryonic germ （EG） cells are cultured pluripotent stem cells derived from the primordial germ cells （PGCs） that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the...Embryonic germ （EG） cells are cultured pluripotent stem cells derived from the primordial germ cells （PGCs） that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22-30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24-28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase （AP）. These cells were able to form embryoid bodies （EBs） in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein （AFP）, s-smooth muscle actin （^-SMA）, and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunoftuorescence for neuronal class III [3-tubulin （Tuj 1）, glial fibrillary protein （GFAP） and galactosylceramidase （GALC）, respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.展开更多
Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variet...Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.展开更多
基金supported by the National Basic Research Program of China(Program 973)(Nos.2009CB941002 and 2011CB944202)the Distinguished Young Scholar Foundation of Heilongjiang Province(No.JC200905)
文摘Embryonic germ （EG） cells are cultured pluripotent stem cells derived from the primordial germ cells （PGCs） that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22-30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24-28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase （AP）. These cells were able to form embryoid bodies （EBs） in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein （AFP）, s-smooth muscle actin （^-SMA）, and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunoftuorescence for neuronal class III [3-tubulin （Tuj 1）, glial fibrillary protein （GFAP） and galactosylceramidase （GALC）, respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.
基金This project was supported by the National Natural Science Foundation of China(No.30370773)National Basic Research Program of China(No.2005CB 121000).
文摘Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.
