Large-Scale Immunopurification of Ribonucleoprotein Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays

2014 ◽  
Vol 2014 (3) ◽  
pp. pdb.prot080705-pdb.prot080705 ◽  
Author(s):  
D. C. Rio
Keyword(s):  
Large Scale ◽  
Tiling Microarrays ◽  
Ribonucleoprotein Complexes

204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes

2019 ◽  
Vol 31 (1) ◽  
pp. 227
Author(s):  
F. L. Ongaratto ◽  
P. Rodriguez-Villamil ◽  
U. Ganbaatar ◽  
C. De Frutos ◽  
S. Solin ◽  
...  
Keyword(s):  
Large Scale ◽  
Gene Editing ◽  
The Other ◽  
Scale Production ◽  
Efficient System ◽  
Ribonucleoprotein Complexes ◽  
Group 4 ◽  
Group 2 ◽  
And Control ◽  
Group 1

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery

Science ◽  
2019 ◽  
Vol 365 (6458) ◽  
pp. 1144-1149 ◽  
Author(s):  
Martin Saurer ◽  
David J. F. Ramrath ◽  
Moritz Niemann ◽  
Salvatore Calderaro ◽  
Céline Prange ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Large Scale ◽  
Small Subunit ◽  
Ribosomal Assembly ◽  
Mitochondrial Ribosomes ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Microscopy ◽  
Number Of Factors ◽  
Cellular Machinery

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo–electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

scholarly journals Generation of mutant pigs by lipofection-mediated genome editing in embryos

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maki Hirata ◽  
Manita Wittayarat ◽  
Zhao Namula ◽  
Quynh Anh Le ◽  
Qingyi Lin ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Large Scale ◽  
Gene Editing ◽  
Time Window ◽  
Mode Of Delivery ◽  
Practical Method ◽  
Ribonucleoprotein Complexes ◽  
Mammalian Embryos ◽  
Specialized Equipment ◽  
Reliable Methods

AbstractThe specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

scholarly journals Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

2020 ◽  
Vol 16 (11) ◽  
pp. e1008293
Author(s):  
Dylan Girodat ◽  
Avik K. Pati ◽  
Daniel S. Terry ◽  
Scott C. Blanchard ◽  
Karissa Y. Sanbonmatsu
Keyword(s):  
Time Scales ◽  
Single Molecule ◽  
Conformational Changes ◽  
Time Resolution ◽  
Large Scale ◽  
Md Simulations ◽  
Orientation Factor ◽  
Ribonucleoprotein Complexes ◽  
Fluorescence Measurements

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

scholarly journals Weak but Uniform Enrichment of the Histone Variant macroH2A1 along the Inactive X Chromosome

2008 ◽  
Vol 29 (1) ◽  
pp. 150-156 ◽  
Author(s):  
Flore Mietton ◽  
Aditya K. Sengupta ◽  
Annie Molla ◽  
Gisele Picchi ◽  
Sophie Barral ◽  
...  
Keyword(s):  
X Chromosome ◽  
Genome Stability ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Chromosome Region ◽  
Histone Variant ◽  
Hek 293 ◽  
Tiling Microarrays ◽  
Green Fluorescent

ABSTRACT We studied the enrichment and distribution of the histone variant mH2A1 in the condensed inactive X (Xi) chromosome. By using highly specific antibodies against mH2A1 and stable HEK 293 cell lines expressing either green fluorescent protein (GFP)-mH2A1 or GFP-H2A, we found that the Xi chromosome contains ∼1.5-fold more mH2A1 than the autosomes. To determine the in vivo distribution of mH2A1 along the X chromosome, we used a native chromatin immunoprecipitation-on-chip technique. DNA isolated from mH2A1-immunoprecipitated nucleosomes from either male or female mouse liver were hybridized to tiling microarrays covering 5 kb around most promoters or the entire X chromosome. The data show that mH2A1 is uniformly distributed across the entire Xi chromosome. Interestingly, a stronger mH2A1 enrichment along the pseudoautosomal X chromosome region was observed in both sexes. Our results indicate a potential role for macroH2A in large-scale chromosome structure and genome stability.

Some comments about observations and image processing of comet 29P/Schwassmann-Wachmann 1

1999 ◽  
Vol 173 ◽  
pp. 243-248
Author(s):  
D. Kubáček ◽  
A. Galád ◽  
A. Pravda
Keyword(s):  
Image Processing ◽  
Large Scale ◽  
Harvard College ◽  
Oak Ridge ◽  
Large Scale Structures ◽  
Short Period Comet ◽  
Short Period ◽  
Scale Structures ◽  
Harvard College Observatory ◽  
Period Comet

AbstractUnusual short-period comet 29P/Schwassmann-Wachmann 1 inspired many observers to explain its unpredictable outbursts. In this paper large scale structures and features from the inner part of the coma in time periods around outbursts are studied. CCD images were taken at Whipple Observatory, Mt. Hopkins, in 1989 and at Astronomical Observatory, Modra, from 1995 to 1998. Photographic plates of the comet were taken at Harvard College Observatory, Oak Ridge, from 1974 to 1982. The latter were digitized at first to apply the same techniques of image processing for optimizing the visibility of features in the coma during outbursts. Outbursts and coma structures show various shapes.

Evolution of Large-Scale Coronal Structures

1994 ◽  
Vol 144 ◽  
pp. 29-33
Author(s):  
P. Ambrož
Keyword(s):  
Magnetic Field ◽  
Field Line ◽  
Large Scale ◽  
Coronal Magnetic Field ◽  
Source Surface ◽  
Solar Cycles ◽  
Characteristic Relationship ◽  
The Magnetic Field ◽  
Coronal Structures

AbstractThe large-scale coronal structures observed during the sporadically visible solar eclipses were compared with the numerically extrapolated field-line structures of coronal magnetic field. A characteristic relationship between the observed structures of coronal plasma and the magnetic field line configurations was determined. The long-term evolution of large scale coronal structures inferred from photospheric magnetic observations in the course of 11- and 22-year solar cycles is described.Some known parameters, such as the source surface radius, or coronal rotation rate are discussed and actually interpreted. A relation between the large-scale photospheric magnetic field evolution and the coronal structure rearrangement is demonstrated.

Large-scale Motion of Solar Filaments

2000 ◽  
Vol 179 ◽  
pp. 205-208
Author(s):  
Pavel Ambrož ◽  
Alfred Schroll
Keyword(s):  
Magnetic Flux ◽  
Proper Motion ◽  
Time Scale ◽  
Large Scale ◽  
Accuracy Of Measurements ◽  
Solar Filaments ◽  
General Movement ◽  
Scale Motion ◽  
Velocity Scatter

AbstractPrecise measurements of heliographic position of solar filaments were used for determination of the proper motion of solar filaments on the time-scale of days. The filaments have a tendency to make a shaking or waving of the external structure and to make a general movement of whole filament body, coinciding with the transport of the magnetic flux in the photosphere. The velocity scatter of individual measured points is about one order higher than the accuracy of measurements.

Some applications of electron beam microanalysis techniques in VLSI process evaluation

1983 ◽  
Vol 41 ◽  
pp. 160-161
Author(s):  
Simon Thomas
Keyword(s):  
Integrated Circuits ◽  
Process Evaluation ◽  
Large Scale ◽  
Technology Development ◽  
Analytical Techniques ◽  
Successful Implementation ◽  
Analysis Of Failures ◽  
Characterization Of Materials ◽  
Circuits Vlsi

Trends in the technology development of very large scale integrated circuits (VLSI) have been in the direction of higher density of components with smaller dimensions. The scaling down of device dimensions has been not only laterally but also in depth. Such efforts in miniaturization bring with them new developments in materials and processing. Successful implementation of these efforts is, to a large extent, dependent on the proper understanding of the material properties, process technologies and reliability issues, through adequate analytical studies. The analytical instrumentation technology has, fortunately, kept pace with the basic requirements of devices with lateral dimensions in the micron/ submicron range and depths of the order of nonometers. Often, newer analytical techniques have emerged or the more conventional techniques have been adapted to meet the more stringent requirements. As such, a variety of analytical techniques are available today to aid an analyst in the efforts of VLSI process evaluation. Generally such analytical efforts are divided into the characterization of materials, evaluation of processing steps and the analysis of failures.

Electron microscopy study of reactively sputter-deposited Ti/N films on silicon

1992 ◽  
Vol 50 (2) ◽  
pp. 1362-1363
Author(s):  
V. C. Kannan ◽  
A. K. Singh ◽  
R. B. Irwin ◽  
S. Chittipeddi ◽  
F. D. Nkansah ◽  
...  
Keyword(s):  
Integrated Circuits ◽  
Large Scale ◽  
Hexagonal Phase ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Process Conditions ◽  
Tin Films ◽  
Wide Range ◽  
Fcc Phase ◽  
Circuits Vlsi

Titanium nitride (TiN) films have historically been used as diffusion barrier between silicon and aluminum, as an adhesion layer for tungsten deposition and as an interconnect material etc. Recently, the role of TiN films as contact barriers in very large scale silicon integrated circuits (VLSI) has been extensively studied. TiN films have resistivities on the order of 20μ Ω-cm which is much lower than that of titanium (nearly 66μ Ω-cm). Deposited TiN films show resistivities which vary from 20 to 100μ Ω-cm depending upon the type of deposition and process conditions. TiNx is known to have a NaCl type crystal structure for a wide range of compositions. Change in color from metallic luster to gold reflects the stabilization of the TiNx (FCC) phase over the close packed Ti(N) hexagonal phase. It was found that TiN (1:1) ideal composition with the FCC (NaCl-type) structure gives the best electrical property.

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