The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.
Cloned animals suffer a wide range of severe fetal and placental malformations. Whether these malformations arise from insufficient epigenetic modifications or mutations has not yet been determined. To address this question, we examined siblings from both cloned XO and XY parents. These parents, which exhibited hypertrophic placentas, increased body weights, and open eyelids at birth, were created from the same ES cell sublines. The siblings from all three cloned pairs showed normal body and placenta weights and no open eyelids at birth. The results clearly showed that the phenotypic abnormalities seen in cloned mice were not transmitted to the progeny, a finding that suggests that abnormalities in cloned mice are responsible for insufficient epigenetic modifications/reprogramming.
In mammals, cloned individuals can be produced from somatic cells. The combined use of gene targeting in embryonic stem cells and cloning contributes to the investigation of gene function in mammals. However, one of the major limitations to cloning is the low viability of cloned embryos, leading typically to high rates of pre- and postnatal death. The present study investigated whether cloning efficiency is influenced by the procedural differences involved in using transfected embryonic stem cells arrested at M phase for cloning by both single and serial transfer. In contrast to a previous study, in which fibroblasts were used, in the present study using embryonic stem cells there was no difference in the rate of production of cloned pups after the use of a single or serial nuclear transfer, although the proportion of blastocysts (70% versus 51%) was significantly higher (P < 0.001) after serial nuclear transfer. After embryo transfer of 445 blastocysts, 218 (49%) implanted and 27 (6% of blastocysts transferred) live pups were born. Of these 27 pups, 23 developed to adults of apparently normal fertility. Of these adults, 39% (n = 9) were derived from targeted embryonic stem cells, which is similar to the proportion of targeted embryonic stem cells in the population used for cloning. This study showed that cloning with embryonic stem cells is a viable procedure resulting in the production of transgenic cloned adults.
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