Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the Mil stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an Mil spindle was present in both pro-MI and MII cytoplasm I h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer

Effects of donor oocytes and culture conditions on development of cloned mice embryos

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 × DBA/2) and B6CBAF1 (C57BL/6 × CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos

Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv × 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines