Document Detail


Generation and characterization of embryonic stem-like cell lines derived from in vitro fertilization Buffalo (Bubalus bubalis) embryos.
MedLine Citation:
PMID:  19144015     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.
Authors:
B Huang; T Li; X-L Wang; T-S Xie; Y-Q Lu; F M da Silva; D-S Shi
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-12-22
Journal Detail:
Title:  Reproduction in domestic animals = Zuchthygiene     Volume:  45     ISSN:  1439-0531     ISO Abbreviation:  Reprod. Domest. Anim.     Publication Date:  2010 Feb 
Date Detail:
Created Date:  2010-02-08     Completed Date:  2010-04-26     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9015668     Medline TA:  Reprod Domest Anim     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  122-8     Citation Subset:  IM    
Affiliation:
Animal Reproduction Institute, Guangxi University, Nanning, Guangxi, PR China. ardsshi@gxu.edu.cn
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / analysis
Animals
Antigens, CD15 / analysis
Antigens, Tumor-Associated, Carbohydrate / analysis
Biological Markers / analysis
Blastocyst / cytology*
Buffaloes / embryology*
Cell Differentiation
Cell Separation / methods,  veterinary
Cells, Cultured
Embryonic Stem Cells / chemistry,  cytology*
Female
Fertilization in Vitro / veterinary*
Homeodomain Proteins / genetics
Male
Morula / cytology*
Octamer Transcription Factor-3 / analysis,  genetics
Pluripotent Stem Cells / chemistry,  cytology
RNA, Messenger / analysis
Reverse Transcriptase Polymerase Chain Reaction / veterinary
SOXB1 Transcription Factors / genetics
Stage-Specific Embryonic Antigens / analysis
Chemical
Reg. No./Substance:
0/Antigens, CD15; 0/Antigens, Tumor-Associated, Carbohydrate; 0/Biological Markers; 0/Homeodomain Proteins; 0/Octamer Transcription Factor-3; 0/RNA, Messenger; 0/SOXB1 Transcription Factors; 0/Stage-Specific Embryonic Antigens; 0/stage-specific embryonic antigen-3; 0/stage-specific embryonic antigen-4; EC 3.1.3.1/Alkaline Phosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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