The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.
Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2-4 and 8-10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated β-galactosidase (SA-β-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-β-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.
Abstract Forkhead box L2 (FOXL2) plays a critical role in the development and function of mammalian ovaries. In fact, the causative effects of FOXL2 misregulations have been identified in many ovarian diseases, such as primary ovarian insufficiency and granulosa cell tumor; however, the mechanism by which FOXL2 expression is regulated is not well studied. Here, we showed that FOXL2 expression in ovarian mural granulosa cells (MGCs) requires stimulation by both oocyte-derived signals and estrogen in mice. In the absence of oocytes or estrogen, expression of FOXL2 and its transcriptional targets, Cyp19a1 and Fst mRNA, in MGCs were significantly decreased. Moreover, expression levels of Sox9 mRNA, but not SOX9 protein, were significantly increased in the FOXL2-reduced MGCs. FOXL2 expression in MGCs was maintained with either oocytes or recombinant proteins of oocyte-derived paracrine factors, BMP15 and GDF9, together with estrogen, and this oocyte effect was abrogated with an ALK5 inhibitor, SB431542. In addition, the FOXL2 level was significantly decreased in MGCs isolated from Bmp15 −/− / Gdf9 +/− mice. Therefore, oocyte, probably with estrogen, plays a critical role in the regulation of FOXL2 expression in mural granulosa cells in mice.
Technologies that prevent housing equipments from the deposition of stains have been developed in our company. Among them, a new technology to prevent a toilet bowl from stains is introduced in this report. The toilet bowl is soiled with two types of stains. The one is the inorganic scale that covers to the dried surface of the toilet bowl, and the other is the organic slime that covers the surface soaked in water. Because the inorganic scale is considered to be caused by adhesion of the silicate in tap water, it could be prevented by coating organic molecule on the bowl surface. On the other side, the organic scale that is considered to be caused by the propagation of bacteria could be solved by antibacterial technology. In this way, different problems of deposited stains must be prevented by different approaches.
Summary Mammals self-regulate their body size throughout development. In the uterus, embryos are properly regulated to be a specific size at birth. Previously, size and cell number in aggregated embryos, which were made from two or more morulae, and half embryos, which were halved at the 2-cell stage, have been analysed in vivo in preimplantation and post-implantation development in mice. Here, we examined whether or not the mouse embryo has the capacity to self-regulate growth using an in vitro culture system. To elucidate embryonic histology, cells were counted in aggregated or half embryos in comparison with control embryos. Both double- and triple-aggregated embryos contained more cells than did control embryos during all culture periods, and the relative growth ratios showed no growth inhibition in an in vitro culture system. Meanwhile, half embryos contained fewer cells than control embryos, but the number grew throughout the culture period. Our data suggest that the growth of aggregated embryos is not affected and continues in an in vitro culture system. On the other hand, the growth of half embryos accelerates and continues in an in vitro culture system. This situation, in turn, implied that post-implantation mouse embryos might have some potential to regulate their own growth and size as seen by using an in vitro culture system without uterus factors. In conclusion, our results indicated that embryos have some ways in which to regulate their own size in mouse early development.
Induction of mouse oocyte meiotic resumption and cumulus expansion in vivo requires a functional EGF network in granulosa cells and paracrine enabling signals from oocytes, yet how these two systems of signaling coordinate in cumulus cells is not understood. Here, we report genetic evidence that oocyte-derived BMP15 and GDF9 promote the expression of functional EGFRs in cumulus cells. The steady-state level of Egfr mRNA was significantly reduced (40% of wildtype levels) in cumulus cells from eCG-primed Bmp15-/- mice, and a more dramatic reduction (20% of the widetype levels) was observed in Bmp15-/- Gdf9+/- double mutant (DM) mice. EGFR protein was also dramatically reduced in cumulus cells of both types of mutant mice. The acute response (activation of MAPK3/1) of DM cumulus cells to a short (30 min) challenge by EGFR ligands (10 ng/ml of EGF and 25 ng/ml of AREG respectively) in vitro, as well as the expression of mRNAs encoding EGF-like peptides, AREG, EREG and BTC in DM cumulus cells at 4 h after administration of hCG in vivo were also dramatically impaired. This indicates that BMP15 and GDF9 promote the expression of functional EGFRs in cumulus cells in vivo. This effect was recapitulated in vitro by removing oocytes from wildtype oocyte-cumulus cells complexes (OCCs). Oocyte removal caused a gradual reduction of Egfr mRNA in cumulus cells: initial significant reduction occurs at 4 h, and further dramatic reduction appears at 8-20 h after oocyte removal. The levels of EGFR protein and activation of MAPK3/1 by EGF measured in cumulus cells at 20 h after oocyte removal were also significantly reduced. The reduction of Egfr mRNA in cumulus cells caused by oocyte removal was prevented by co-culture with wildtype, but not fully grown Bmp15-/- or DM oocytes or wildtype growing oocytes from late secondary follicles. EGF-induced expression of mRNAs for EGF-like peptides in cumulus cells was also impaired by immediate removal of oocytes. Although an increase in the levels of Areg and Ereg mRNAs occurred at 2 h in oocyte-removed cumulus cells, the levels of the mRNAs for all three peptides was significantly reduced at 4 h compared to cumulus cells of intact COCs. Furthermore, treatment of wildtype OCCs with a SMAD2/3 inhibitor (5 μM SB43152), but not a SMAD3 inhibitor (20 μM SIS), resulted in dramatic reduction of Egfr mRNA in cumulus cells. We conclude that oocytes license the induction of OCC maturation by promoting the expression of functional EGFRs in cumulus cells, and a SMAD2-dependent pathway is involved in this process. BMP15 and GDF9 are prominent candidates for the oocyte-derived paracrine licensing factors. Supported by HD23839 (Y-QS, KS, JJE), HD21970 (KS and JJE).
Zinc finger nucleases (ZFN), which are artificial restriction enzymes consisting of an engineered zinc-finger domain (ZF) and an endonuclease domain, can be used for the induction of site-directed mutation and the efficient generation of gene knockout animals. However, the repeated construction of various ZFN sequences is both expensive and time consuming. In this study, we attempted to establish a novel method for inexpensive and rapid ZFN construction. First, we constructed ZFN against mouse Rosa26 and original mouse Gli3 gene loci using short PCR primer sets (>30 bp), which contained 21 bp of the ZF recognition helix for a specific DNA triplet. We prepared 18 sets of such primers and PCR was performed using one of these primer sets and the partial ZF sequence as a template, which was obtained from the first to second DNA recognition helix of mouse Zif268. The PCR products were joined by overlap-PCR and nested PCR, and then inserted into a vector coding the endonuclease domain of FokI nuclease. By these steps, we successfully synthesised intended ZFN vectors containing 4 to 6 fingers. Next, we evaluated the functions of constructed ZFN. The mRNA of constructed ZFN were transcribed in vitro and injected into the cytoplasm of C57BL/6N zygotes. After 24 h of culture, 2-cell stage embryos were subjected to genomic PCR of the target locus, and the PCR products were directly sequenced. When ZFN mRNA for mouse Rosa26 was injected, 3- to 146-bp deletions were detected in 92.8% of injected embryos. This result was almost the same as previously reported for ZFN, indicating that our novel construction method can synthesise functional ZFN, which work as a site-directed nuclease, and that efficiency was comparable with those constructed by conventional PCR methods using long oligonucleotide sets (60 bp).
