Document Detail

Generation of buffalo (Bubalus bubalis) transgenic chimeric and nuclear transfer embryos using embryonic germ-like cells expressing enhanced green fluorescent protein.
MedLine Citation:
PMID:  19144016     Owner:  NLM     Status:  MEDLINE    
The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)-like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG-like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE-EGFP-IRES-Neo-dNdB) using Lipofectamine 2000 and selected by culturing in 200 microg/ml G418 for 6-8 days. G418 resistant fibroblasts and EG-like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8-16 cells embryos were injected with EG-like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG-like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG-like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG-like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG-like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG-like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.
B Huang; K Cui; T Li; X Wang; F Lu; Q Liu; F M da Silva; D 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:  103-8     Citation Subset:  IM    
Animal Reproduction Institute, Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Guangxi, China.
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MeSH Terms
Blastocyst / chemistry,  physiology
Buffaloes / embryology*
Chimera / genetics*
Embryo Culture Techniques / veterinary
Fertilization in Vitro / veterinary
Fibroblasts / ultrastructure
Gene Expression
Germ Cells / ultrastructure*
Green Fluorescent Proteins / genetics*
Nuclear Transfer Techniques / veterinary*
Oocytes / ultrastructure
Organisms, Genetically Modified / genetics*
Transfection / veterinary
Reg. No./Substance:
0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins

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