title: Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.
abstract: 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.
authors.affiliation: Bundangcheil Women's Clinic, Sung-nam, Korea; Bio-Organ Research Center, Konkuk University, Seoul, Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Ju
authors.forename: Chun Young
authors.initials: CY
authors.lastname: Park
authors.forename: Mukesh Kumar
authors.initials: MK
authors.lastname: Gupta
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Uhm
authors.forename: Eun Chan
authors.initials: EC
authors.lastname: Paik
authors.forename: Zae Young
authors.initials: ZY
authors.lastname: Ryoo
authors.forename: Youl Hee
authors.initials: YH
authors.lastname: Cho
authors.forename: Kil Saeng
authors.initials: KS
authors.lastname: Chung
authors.forename: Hoon Taek
authors.initials: HT
authors.lastname: Lee
mesh_terms: 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*
chemical_list: D010919:Placebos