Viable transgenic goats derived from skin cells.
The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.
Behboodi
Esmail
E
GTC-Biotherapeutics Inc., 5 Mountain Road, Framingham, MA, 01701, USA.
Memili
Erdogan
E
Melican
David T
DT
Destrempes
Margaret M
MM
Overton
Susan A
SA
Williams
Jennifer L
JL
Flanagan
Peter A
PA
Butler
Robin E
RE
Liem
Hetty
H
Chen
Li How
LH
Meade
Harry M
HM
Gavin
William G
WG
Echelard
Yann
Y
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000502:physiology; D002453:Cell Cycle; D002455:Cell Division; D019976:Cloning, Organism / Q000379:methods*; D004624:Embryo Transfer; D047108:Embryonic Development / Q000502:physiology; D005187:Fallopian Tubes / Q000166:cytology / Q000502:physiology; D005260:Female; D047109:Fetal Development / Q000502:physiology; D006041:Goats / Q000196:embryology*; D017404:In Situ Hybridization, Fluorescence; D009028:Morula / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D012867:Skin / Q000166:cytology*