Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer.
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, 73.8 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.
Gong
Guochun
G
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, China.
Dai
Yunping
Y
Fan
Baoliang
B
Zhu
Huabing
H
Zhu
Shien
S
Wang
Haiping
H
Wang
Lili
L
Tang
Bo
B
Li
Rong
R
Wan
Rong
R
Liu
Ying
Y
Huang
Yinhua
Y
Zhang
Lei
L
Sun
Xiuzhu
X
Li
Ning
N
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000378:metabolism*; D015139:Blotting, Southern; D002417:Cattle; D019976:Cloning, Organism; D015925:Cryopreservation; D017930:Genes, Reporter; D018895:Microsatellite Repeats; D053652:Nuclear Transfer Techniques; D016133:Polymerase Chain Reaction