Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts.
We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new "peeling" method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin beta3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.
Kwon
Dae Kee
DK
Department of Theriogenology and Biotechnology, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea.
Hong
So Gun
SG
Park
Hee Jung
HJ
Kang
Jung Taek
JT
Koo
Ok Jae
OJ
Lee
Byeong Chun
BC
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D005855:Germ Layers / Q000166:cytology*; D014404:Tubulin / Q000378:metabolism
D014404:Tubulin; D000469:Alkaline Phosphatase