The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by ...using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press. Here, we found that 5–50
nM TSA-treatment for 10
h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.
Bone morphogenetic proteins (BMPs) play pleiotropic roles during embryonic development as well as throughout life. Recent genetic approaches especially using the mouse gene knockout system revealed ...that BMP signaling is greatly involved in early embryonic patterning, which is a dynamic event to establish three-dimensional polarities. The purpose of this review is to describe the diverse function of BMPs through different receptor signaling systems during embryonic patterning including gastrulation and establishment of the left–right asymmetry.
The Telescope Array (TA) observatory utilizes fluorescence detectors and surface detectors (SDs) to observe air showers produced by ultra high energy cosmic rays in Earth's atmosphere. Cosmic-ray ...events observed in this way are termed hybrid data. The depth of air shower maximum is related to the mass of the primary particle that generates the shower. This paper reports on shower maxima data collected over 8.5 yr using the Black Rock Mesa and Long Ridge fluorescence detectors in conjunction with the array of SDs. We compare the means and standard deviations of the observed X max distributions with Monte Carlo X max distributions of unmixed protons, helium, nitrogen, and iron, all generated using the QGSJet II-04 hadronic model. We also perform an unbinned maximum likelihood test of the observed data, which is subjected to variable systematic shifting of the data X max distributions to allow us to test the full distributions, and compare them to the Monte Carlo to see which elements are not compatible with the observed data. For all energy bins, QGSJet II-04 protons are found to be compatible with TA hybrid data at the 95% confidence level after some systematic X max shifting of the data. Three other QGSJet II-04 elements are found to be compatible using the same test procedure in an energy range limited to the highest energies where data statistics are sparse.
It has now been 13 years since the first cloned mammal Dolly the sheep was generated from somatic cells using nuclear transfer (SCNT). Since then, this technique has been considered an important tool ...not only for animal reproduction but also for regenerative medicine. However, the success rate is still very low and the mechanisms involved in genomic reprogramming are not yet clear. Moreover, the NT technique requires donated fresh oocyte, which raises ethical problems for production of human cloned embryo. For this reason, the use of induced pluripotent stem cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, we believe that the NT approach remains the only valid way for the study of reproduction and basic biology. For example, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, and it can generate offspring from a single cell or even a frozen dead body. Thanks to much hard work by many groups, cloning success rates are increasing slightly year by year, and NT cloning is now becoming a more applicable method. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.
The electron transfer system of the biphenyl dioxygenase BphA, which is derived from
Acidovorax sp. (formally
Pseudomonas sp.) strain KKS102, is composed of an FAD-containing NADH-ferredoxin ...reductase (BphA4) and a Rieske-type 2Fe-2S ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the 2Fe-2S cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3–BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.
Although round spermatid injection can be used to create progeny for males who do not produce mature sperm, the rate of successful embryogenesis after such procedures is significantly lower than that ...for similar procedures using mature spermatozoa. The mechanisms underlying this difference are unknown. In this study, we demonstrate that, unlike the normal paternal genome, the paternal zygotic genome derived from a round spermatid is highly remethylated before first mitosis after demethylation. Genomes from elongated spermatids exhibited an intermediate level of DNA methylation, between those of round spermatids and mature spermatozoa, suggesting that the male germ cell acquires the ability to maintain its undermethylated state in the paternal zygotic genome during this phase of spermiogenesis. In addition, treatment of zygotes with trichostatin A led to a significant reduction in DNA methylation, specifically in the spermatid-derived paternal genome, except for the pericentromeric regions enriched by trimethylation of Lys9 of histone H3. These data provide insight into epigenetic errors that may be associated with the poor development of embryos generated from immature spermatozoa.
Without using sperm, artificial oocyte activation is essential for current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has ...been widely used as an activator of oocytes especially in the mouse, by which efficient oocyte activation requires Casup(2+)-free medium. In this study, we examined whether Srsup(2+) can efficiently activate oocytes in Casup(2+)-containing culture media when calcium is chelated. Ethylene glycol-bis (beta-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) was added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because it preferentially binds Casup(2+) rather than Srsup(2+) We found that treatment with 5 mM Srsup(2+) and 2 mM EGTA left fewer than 1% of oocytes at the MII stage, which is comparable to that of Casup(2+)-free medium. As a result, addition of 2 mM EGTA along with 5 mM Srsup(2+) in either CZB, M16 or KSOM made more than 80% of available activated oocytes, which was comparable to or better than 72% in a Casup(2+)-free Srsup(2+) medium, since EGTA-Srsup(2+) activation led to significantly less oocyte degeneration than Casup(2+)-free Srsup(2+) activation. Furthermore, we demonstrated that this activation method can support the birth of cloned embryos. Thus, addition of EGTA to typical Casup(2+)-containing culture media can easily produce activation media that does not interfere with embryonic development.
Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a ...decade. Further in mice, the 'clonable' strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally 'unclonable' strains.
Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative
medicine. However, it is still unclear whether any individual or ...cell type can be used to generate such lines. Here, we tested
seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing
nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from
F 1 mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells;
however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on
preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All
mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission
in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos.
Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and
cell types of both sexes, even though it may be more difficult to generate clones directly.
Abstract
Nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse strains and cell types
from both sexes, even though it may be almost impossible to generate clones directly
Cloning mammals by somatic cell nuclear transfer (SCNT) has become an established procedure, but the success rate remains low and gene expression abnormalities are also observed. In addition, SCNT ...pups exhibited an abnormal gene expression profile with a high degree of heterogeneity among individuals. Recently, we reported that somatic clones treated with trichostatin A (TSA) exhibited a significantly improved success rate, probably due to its effects on chromatin remodeling and histone modification in early embryos. Here we show that the TSA treatment also improves the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA clone pups decreased to half of the conventional SCNT pups, and the variation among individuals observed in the SCNT pups was also reduced to the level of the pups produced by the intracytoplasmic sperm injection (ICSI) method. Interestingly, the total gene expression profile of the TSA clones came to resemble that of the ICSI pups.
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