Generation of Buffalo (Bubalus bubalis) Transgenic Chimeric and Nuclear Transfer Embryos Using Embryonic Germ-Like Cells Expressing Enhanced Green Fluorescent Protein
Agricultura e Produção Animal
Ben Huang, Kuiqing Cui, Tisan Xie, Tong Li, F. Moreira da Silva and Deshun Shi. 2008. Generation of buffalo (Bubalus bubalis) transgenic chimeric and nuclear transfer embryos using embryonic germ like cells transfected with green fluorescent protein gene. Reproduction in Domestic Animals (Epub ahead of print).
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 LipofectamineTM 2000 and selected by culturing in 200 μg/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
Quinta, 31 Julho, 2008
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