Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer

文献类型： 外文期刊

第一作者： Gong, GC

作者： Gong, GC;Dai, YP;Fan, BL;Zhu, HB;Zhu, S;Wang, HP;Wang, LL;Tang, B;Li, R;Wan, R;Liu, Y;Huang, YH;Zhang, L;Sun, XZ;Li, N

作者机构：

关键词： Nuclear transfer;Enhanced green fluorescent protein;Transgene;Fetal fibroblasts;Cloned pigs;Enucleated oocytes;Granulosa-cells;Mouse embryos;Capra-hircus;Cryopreservation;Vitrification;Adult;Sheep

期刊名称：MOLECULAR REPRODUCTION AND DEVELOPMENT （ 影响因子：2.609； 五年影响因子：3.47 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8% respectively) or transfected FOECs (TFOECs, 73. and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP-embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully.

分类号： Q7

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