Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein.
This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.
Lee
Gab Sang
GS
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Sillim-Dong, Kwanak-ku, Seoul 151-742, South Korea.
Kim
Hye Soo
HS
Hyun
Sang Hwan
SH
Lee
So Hyun
SH
Jeon
Hyun Yong
HY
Nam
Dong Hyun
DH
Jeong
Yeon Woo
YW
Kim
Sue
S
Kim
Ji Hye
JH
Han
Jae Yong
JY
Ahn
Curie
C
Kang
Sung Keun
SK
Lee
Byeong Chun
BC
Hwang
Woo Suk
WS
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001755:Blastocyst / Q000502:physiology; D002118:Calcium / Q000032:analysis; D002459:Cell Fusion; D002460:Cell Line; D019976:Cloning, Organism / Q000379:methods*; D004558:Electric Stimulation; D004624:Embryo Transfer; D047108:Embryonic Development; D004921:Erythropoietin / Q000235:genetics; D005260:Female; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013552:Swine / Q000235:genetics*; D014162:Transfection
D004921:Erythropoietin; D049452:Green Fluorescent Proteins; D002118:Calcium