Establishment of Embryonic Stem cell Lines from Parthenogenetic and Cloned Mice Embryos Produced by Nuclear Transfer

Abstract

This study was designed to establish the ES cell line from parthenogenetic as well as cloned mouse blastocysts produced by the nuclear transfer of cumulus cell nuclei. Metaphase II (MII) stage oocytes were recovered from superovulated 129B6 F1 (C57BL/6 X 129P3/J) hybrid strain mice and were nuclear transferred with cumulus cell nuclei, or they were parthenogenetically activated by SrCl2 and cytochalasin B after sham-enucleation. Nuclear transferred oocytes showed a significantly lower morula/blastocyst development rate of 19.0 against 100% in the sham-enucleated group (P<0.05). A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the NT embryos (60.3 and 29.3%). Two ES cell lines could be established from the 11 NT and 13 sham-enucleated, parthenogenetically activated mouse embryos. The cell colonies displayed the typical morphology of mice ES cells. The ES cell lines could be successfully maintained with undifferentiated morphology after continuous proliferation for more than 95 passages yet maintaining normal karyotype. These cells also expressed mice ES cell markers such as Oct 4 and SSEA-1. RT-PCR further confirmed the Oct 4 expression in the ntES cells. The clonal origin of the ntES cell line and the parthenogenetic ES cell lines were confirmed by PCR analysis of the polymorphic markers. Thus, the present study reports on the successful establishment of mice ES cells from oocyte nuclear transfer with cumulus cell nuclei, as well as from parthenogenetically activated oocytes.