9 research outputs found
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U.S./Russian lab-to-lab materials protection, control and accounting program efforts at the Institute of Inorganic Materials. Revision 1
The All-Russian Scientific Research Institute of Inorganic Materials (VNIINM) performs research in nuclear power reactor fuel, spent fuel reprocessing and waste management, materials science of fissionable and reactor structural materials, metallurgy, superconducting materials, and analytical sciences. VNIINM supports the Ministry of Atomic Energy of the Russian Federation (MINATOM) in technologies for fabrication and processing of nuclear fuel. As a participant in the US/Russian Lab-to-Lab nuclear materials protection, control and accounting (MPC and A) program, VNIINM is providing support for measurements of nuclear materials in bulk forms by developing specifications, test and evaluation, certification, and implementation of measurement methods for such materials. In 1996, VNIINM will be working with Brookhaven staff in developing and documenting material control and accounting requirements for nuclear materials in bulk form, Livermore and Los Alamos staff in testing and evaluating gamma-ray spectrometry methods for bulk materials, Los Alamos staff in test and evaluation of neutron-coincidence counting techniques, Oak Ridge staff in accounting of bulk materials with process instrumentation, and Pacific Northwest staff on automating VNIINM`s coulometric titration system. In addition, VNIINM will develop a computerized accounting system for nuclear material within VNIINM and their storage facility. The paper will describe the status of this work and anticipated progress in 1996
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RUSSIAN NDA REFERENCE MATERIALS WORKING GROUP (RWG): ITS CREATION, MISSION, GOALS, AND STATUS
Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase
<div><p>To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.</p></div
Target dominant upregulation of DNA methylation by TALMaj-SssI expression in mouse embryos.
<p>(A) Bisulfite sequencing of TALMaj-SssI-expressing embryos. Methylation of major and minor satellite CpGs was evaluated in TALMaj-SssI embryos. TALMaj-SssI was expressed at 3 levels (low, middle, and high, from left to right) by injecting various concentrations (2, 10, and 50 ng/μL, respectively) of RNAs encoding TALMaj-SssI (WT or T313D) into fertilized embryos. Mock control indicates normal fertilized embryos without injection. Embryos were recovered 3 days after RNA injection. (B) DNA methylation of IAP and Line1 was evaluated in TALMaj-SssI-expressing embryos. Embryos expressing the middle level (10 ng/μL RNA injection) of TALMaj-SssI were evaluated. Mock control indicates normal fertilized embryos without injection. Data are expressed as the percentage of methylated CpG sites relative to all CpG sites. Asterisks indicate significant differences between the mock control and tested group by the Mann–Whitney <i>U</i> test.</p
Upregulation of major satellite DNA methylation does not hamper chromosome segregation in preimplantation embryos.
<p>(A) Chromosome segregation patterns observed in embryos expressing the TALMaj-SssI (WT) fusion protein. Snapshots of the 2-cell to 4-cell transition are indicated. Embryos were injected with RNAs encoding mCherry-MBD-NLS (5 ng/μL), H2B-EGFP (10 ng/μL) and TALMaj-SssI (WT or T313D; 10 ng/μL) and we observed EGFP fluorescence during the 1-cell to 8-cell stages by live-cell imaging. Embryos were classified into two groups: abnormal chromosome segregation (ACS) and normal chromosome segregation (NCS). Arrows indicate lagging chromosomes. Scale bar = 20 μm. (B) Frequency of ACS in embryos expressing TALMaj-SssI. These were classified as having 1–2 ACS, 2–4 ACS, or 4–8 ACS, based on the timing of ACS at the 1-cell to 2-cell, 2-cell to 4-cell and 4-cell to 8-cell stages, respectively. In all, 29 WT and 24 T313D embryos were assessed for ACS analysis. The ACS frequencies in these three groups were compared with embryos expressing the WT or T313D proteins by chi-squared test. No statistically significant difference was observed.</p
Upregulation of pericentromeric DNA methylation by TALMaj-SssI expression in mouse embryos.
<p>(A) Live-cell imaging of DNA methylation in TALMaj-SssI expressing embryos during first mitosis. Fertilized embryos were injected with RNAs encoding TALMaj-SssI, mCherry-MBD-NLS (Red) and H2B-EGFP (Green). Enzymatically active form of CpG methyltransferase SssI (WT) and inactivated SssI (T313D) was tested in this experiment. Mock indicates no injection of TALMaj-SssI. Scale bar represents 20μm. (B) Time-lapse analysis of DNA methylation during the first mitosis. Time-lapse observations were made of embryos expressing both mCherry-MBD-NLS and TALMaj-SssI (WT, T313D, or mock injection). Snapshots indicate DNA methylation (MBD) at each mitotic stage. Scale bar represents 20 μm. (C) Quantification of DNA methylation in embryos expressing TALMaj-SssI. MBD fluorescence of chromosomes was quantified at each mitotic stage. Each data point represents the analysis of 3 embryos. Asterisks indicate significant differences by two-way ANOVA (<i>p</i><0.05). (D) Live-cell imaging of DNA methylation status in TALMaj-SssI expressing 2-cell embryos. Enzymatically active (WT) and inactive (T313D) TALMaj-SssI was expressed in 2-cell embryos with mCherry-MBD-NLS (Red) and H2B-EGFP (Green). 2-cell images were captured about 4–5 h after 2-cell division. Scale bar represents 20μm. (E) Quantification of the heterochromatin index (Ueda et al, 2014) in embryos expressing TALMaj-SssI WT, T313D, and mCherry-MBD-NLS. Fluorescent signal of mCherry-MBD-NLS was evaluated in 2-cell embryos obtained 17 h after 2-cell division. Asterisks indicate significant difference by Mann-Whitney <i>U</i> test (<i>p</i><0.05).</p
Confirmation of target-preferential CpG methylation activity of TALMaj-SssI.
<p>(A) Structure of major satellite recognition TALE and bacterial CpG methyltransferase, SssI, fusion protein. Major satellite recognition TALE, originally designed by Miyanari et al., were fused with 3Ă—FLAG or FLAG-HA that was tagged enzymatically active (WT) or inactive (T313D) SssI DNA methyltransferase. (B) TALE recognition sequence of the major satellite repeat. Red character indicated TALE recognition 15 nucleotides in the major satellite sequences (GenBank: EF028077). (C) TALMaj-SssI localization at pericentromere. Immunostaining of FLAG-tagged TALMaj-SssI (WT) expressing mouse C3H10T1/2 cultured cells. Cells were transfected TALMaj-SssI-3Ă—FLAG expressing plasmid together with EGFP-CENPC-expressing plasmid. Cells were fixed 48h after transfection and then stained with anti-FLAG antibody. Enlarged images of a single heterochromatin focus in nucleus are shown in the inset in the upper panel. The bottom panel indicates 3D reconstitution images of the staining data. The single heterochromatin focus within the square is enlarged in the right panel. Arrows indicate CENPC which locates around FLAG signal. Arrowheads indicate CENPC which shows slight overlap with FLAG signals. Red: FLAG, Green: EGFP-CENPC, Blue: DAPI. Scale bar represents 20ÎĽm. (D) Upregulation of pericentromeric DNA methylation by TALMaj-SssI expression. TALMaj-SssI (mock vector, WT, or T313D), mCherry-MBD-NLS, and EGFP-CENPC were introduced into Dnmt TKO ES cells. DNA methylation, kinetochores, and DNA are shown by mCherry-MBD-NLS (red), EGFP-CENPC (green), and DAPI (blue), respectively. Arrows indicate induced DNA methylation in DAPI-dense heterochromatin regions. Scale bar represents 20 ÎĽm. (E) ChIP-qPCR analysis of TALMaj-SssI-stably expressing Dnmt TKO ES cells. Localization of TALMaj-SssI (T313D)-3Ă—FLAG was analyzed by ChIP-qPCR with anti-FLAG antibody. Untransfected ES cells were used as the control. Asterisks indicate significant differences by two-way ANOVA (<i>p</i><0.05).</p
Target dominant upregulation of DNA methylation by dCas9-SssI expression in mouse embryos.
<p>(A) Live-cell imaging of DNA methylation in dCas9-SssI expressing 2-cell embryos. Fertilized embryos were injected with RNAs encoding dCas9-SssI, major satellite gRNA, mCherry-MBD-NLS and H2B-EGFP (not shown). Expression level was controlled by adjusting concentration of RNA encoding dCas9-SssI (60ng/μL) and gRNA (60ng/μL). MBD fluorescent images were captured about 4–5 h after 2-cell division. Enzymatically active dCas9-SssI (WT) and inactive dCas9-SssI (T313D) were evaluated. Scale bar represents 20μm. (B) Quantification of the heterochromatin index of embryos expressing dCas9-SssI and mCherry-MBD-NLS and H2B-EGFP. Fluorescent signal of mCherry-MBD-NLS was evaluated with 2-cell embryos obtained 17 h after 2-cell division. Asterisks indicate significant difference by Mann-Whitney <i>U</i> test (<i>p</i><0.05).</p