Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.
OBJECTIVE
To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice.


METHODS
Randomized, prospective study.


METHODS
Hospital-based assisted reproductive technology laboratory.


METHODS
F(1) (C57BL/6 x 129P3/J) mice.


METHODS
Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure.


METHODS
Embryogenesis and embryonic stem cell establishment.


RESULTS
The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent.


CONCLUSIONS
We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.
Ju
Jin Young
JY
Bundangcheil Women's Clinic, Sung-nam, Korea; Bio-Organ Research Center, Konkuk University, Seoul, Korea.
Park
Chun Young
CY
Gupta
Mukesh Kumar
MK
Uhm
Sang Jun
SJ
Paik
Eun Chan
EC
Ryoo
Zae Young
ZY
Cho
Youl Hee
YH
Chung
Kil Saeng
KS
Lee
Hoon Taek
HT
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005838:Genotype; D006822:Hybrid Cells / Q000166:cytology; D007621:Karyotyping; D051379:Mice; D008810:Mice, Inbred C57BL; D008846:Micromanipulation; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D009866:Oogenesis / Q000502:physiology; D010312:Parthenogenesis; D010919:Placebos; D011897:Random Allocation; D013234:Stem Cells / Q000166:cytology*
D010919:Placebos