42 research outputs found
Development of mouse embryos with introduced RNAs and ssODN using the TAKE method.
<p><sup>a</sup>Calculated from the number of embryos examined.</p><p><sup>b</sup>Calculated from the number of embryos developed to two cells.</p><p><sup>c</sup>Calculated from the number of male and female mice.</p><p>Significant differences at <i>P</i> < 0.05; a <i>vs</i>. b, c <i>vs</i>. d, e <i>vs</i>. f.</p><p>Development of mouse embryos with introduced RNAs and ssODN using the TAKE method.</p
Development of rat embryos with introduced RNA and ssODN using the TAKE method.
<p><sup>a</sup>Calculated from the number of embryos examined.</p><p><sup>b</sup>Calculated from the number of embryos developed to two cells.</p><p><sup>c</sup>Calculated from the number of male and female rats.</p><p>Significant differences at <i>P</i> < 0.05; a <i>vs</i>. b, c <i>vs</i>. d.</p><p>Development of rat embryos with introduced RNA and ssODN using the TAKE method.</p
<i>Brca2<sup>(p.T1942fs/+)</sup></i> dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells
Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8–32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.</p
Notochord and floor plate patterning in <i>Oune/+</i> embryos.
<p><i>In situ</i> hybridization analysis was performed on transverse sections through the trunk of E12.5 embryos using RNA probes for <i>Brachyury</i> (A, B), a notochord marker, and <i>Foxa2</i> (C, D), a floor plate marker. No expression change was observed between <i>+/+</i> and <i>Oune/+</i> embryos. Notochord (NC) is indicated by arrowheads and floor plate (FP) is enclosed by yellow lines. Scale bar, 200 μm.</p
Electrophoretic mobility shift assay (EMSA) of <i>Tbx6</i> and <i>Tbx6</i><sup><i>Oune</i></sup>.
<p>(A) The <i>Tbx6</i><sup><i>Oune</i></sup> allele did not influence the DNA binding ability of the T-box binding consensus sequences. <i>Tbx6</i> and <i>Tbx6</i><sup><i>Oune</i></sup> (Tbx6 mut) showed no difference in binding ability to the Tbind probe, two T-box gene binding sites, and to the Tbind-half probe, a single binding site, judged from intensity of shifted bands. Free binding probes were shown at the bottom. (B) DNA binding ability of mutant Tbx6 was not changed. The Tbind probe concentration was diluted to one sixteenth, but no difference was detected between Tbx6-wt and mut. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p
Translational efficiency and transcription activation ability of <i>Tbx6</i> proteins.
<p>(A) Western blotting analysis of S protein-tagged mTbx6 proteins. Cell lysate from transfectant of the <i>mTbx6</i> and <i>mTbx6</i><sup><i>Oune</i></sup> expression constructs (Tbx6 and Tbx6 mut, respectively), in which the coding region of wild type and <i>Oune</i> mutant <i>mTbx6</i> are tagged with partial S protein sequences in N-terminus, was used with anti-S protein (the upper panel) and anti-USF2 (the lower panel) antibodies. Signals of S protein tagged mTbx6 are indicated by the arrow. (B) <i>In vitro</i> translation assays for mTbx6 and mTbx6 mut. Difference of translational efficiency between wild type and mutant mTbx6 was not observed. The S-tag mTbx6 constructs showed multiple translational initiations (tagged protein: asterisk). (C) Transcriptional activation properties of <i>Tbx6</i> and <i>Tbx6</i><sup><i>Oune</i></sup> using a <i>Mesp2</i> promoter-luciferase reporter construct. <i>rTbx6</i><sup><i>Oune</i></sup> activates transcription less effectively than <i>rTbx6</i> when a Notch intracellular domain (NICD) expression construct was cotransfected into C2C12 cultured cells. (D) Transcriptional activation properties of a mixture of <i>Tbx6</i> and <i>Tbx6</i><sup><i>Oune</i></sup> constructs. Half-and-half of rTbx6 and rTbx6 mut constructs with NICD showed intermediate levels of luciferase activities. Assays were performed in triplicate. One-way analysis of variance was performed on data from all experiments, and significance was determined using Turkey's post hoc test. ns, not significant. mTbx6 and rTbx6 represents mouse and rat Tbx6, respectively.</p
Altered expression of Notch pathway components in <i>Oune/+</i> embryos.
<p>Whole mount <i>in situ</i> hybridization with rat <i>Tbx6</i> (A, B), <i>Mesp2</i> (C, D), and <i>Dll1</i> (E, F) probes was performed using E12.5 +/+ and <i>Oune/+</i> embryos. Obvious changes of expression patterns and intensity were not observed. Red boxes (A, B) and arrowheads (C, D) indicate <i>Tbx6</i> expression in tail bud and <i>Mesp2</i> expression in presomitic mesoderm, respectively. The contiguous expression of <i>Dll1</i> in the presomitic mesoderm (bars, E, F) appear extended anteriorly in heterozygous mutant embryos. Arrows indicates expression borders between somites and presomitic mesoderm. Note that low levels of <i>Dll1</i> expression were observed in mutants. Original magnification: 20x (A, B); 90x (C, D); 40x (E-F).</p
Somite pattering in <i>Oune/+</i> embryos.
<p>Sagittal sections of E14.5 +/+ and <i>Oune/+</i> embryos were used for hematoxylin and eosin staining (A, B) and <i>in situ</i> hybridization with various somite markers, <i>Pax1</i> (C-H), <i>Uncx4</i>.<i>1</i> (I-N), and <i>Dll1</i> (O-T). Incomplete somite patterning in the anterior region of <i>Oune/+</i> embryos was observed (B, box a). In the same embryo, somites in the trunk and posterior regions were morphologically normal (B, boxes t and p). In <i>Oune/+</i> embryos, <i>Pax1</i> expression was decreased in the anterior region (F), and somites were dislocated in the posterior region (H). Signals of markers for the caudal half of somites, <i>Uncx4</i>.<i>1</i> and <i>Dll1</i>, were reduced in the anterior and posterior regions of <i>Oune/+</i> embryos (L and N: <i>Uncx4</i>.<i>1</i>; R and T: <i>Dll1</i>). Scale bar, 200 μm.</p
Morphological abnormalities of vertebral column in ENU-induced <i>Oune</i> rats.
<p>(A, B) Perinatal and newborn offspring derived from (<i>Oune/+</i> x <i>+/+</i>) mating pairs. <i>Oune</i> rats were distinguished from their siblings because of kinky tails (yellow bar and arrowheads). (C-F) Ventral view of axial skeletons of newborn wild type and <i>Oune/+</i> siblings. In the cervical and thoracic region, <i>Oune/+</i> rats showed loss and malformations of vertebrae (D, boxes). In the lumbar and sacral region of <i>Oune/+</i> animals, vertebrae were malformed and laterally bent (F, asterisks). An extra lumbar vertebra was frequently observed (F, L7). (G-J) Ventral view of axial skeletons of E15.5 <i>Oune</i> siblings. Wild type embryos showed ordered thoracic vertebral blocks along the anterior-posterior axis (G, bars). In <i>Oune/Oune</i> embryos, vertebral blocks were located along two different axes (H, I bars) with loss of rib formation. Original magnification: 12.5x (C, D); 10x (E, F); 32x (G-I).</p
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Additional file 18: of CLICK: one-step generation of conditional knockout mice
Table S4. Primer sets used for PCR and sequencing analysis in this study. (XLSX 10 kb