Construction of mutant library v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Target Gene ◽  
High Efficiency ◽  
False Positives ◽  
Functional Screening ◽  
Mutant Library

This protocol is used to construct mutant library of target gene with high efficiency and low false positives/negatives rate after subsequent functional screening.

Construction of Mutant Library v1

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Target Gene ◽  
High Efficiency ◽  
False Positives ◽  
Functional Screening ◽  
Mutant Library

This protocol is used to construct mutant library of target gene with high efficiency and low false positives/negatives rate after subsequent functional screening.

scholarly journals Fungal Functional Genomics: Tunable Knockout-Knock-in Expression and Tagging Strategies

2009 ◽  
Vol 8 (5) ◽  
pp. 800-804 ◽  
Author(s):  
Luis F. Larrondo ◽  
Hildur V. Colot ◽  
Christopher L. Baker ◽  
Jennifer J. Loros ◽  
Jay C. Dunlap
Keyword(s):  
Functional Genomics ◽  
Target Gene ◽  
High Efficiency ◽  
Expression System ◽  
Gene Replacement ◽  
Fungal Species ◽  
Homologous Gene ◽  
Molecular Tools ◽  
Phenotypic Information ◽  
Endogenous Loci

ABSTRACT Strategies for promoting high-efficiency homologous gene replacement have been developed and adopted for many filamentous fungal species. The next generation of analysis requires the ability to manipulate gene expression and to tag genes expressed from their endogenous loci. Here we present a suite of molecular tools that provide versatile solutions for fungal high-throughput functional genomics studies based on locus-specific modification of any target gene. Additionally, case studies illustrate caveats to presumed overexpression constructs. A tunable expression system and different tagging strategies can provide valuable phenotypic information for uncharacterized genes and facilitate the analysis of essential loci, an emerging problem in systematic deletion studies of haploid organisms.

scholarly journals A Semi-Quantitative Bulked Segregant Analysis Preliminarily Localizes a Maize Male-Sterility Gene

2021 ◽  
Vol 25 (03) ◽  
pp. 659-666
Author(s):  
Mingbo Wu
Keyword(s):  
Male Sterility ◽  
Target Gene ◽  
Dna Analysis ◽  
High Efficiency ◽  
Bulked Segregant Analysis ◽  
Mutant Gene ◽  
Polymorphic Markers ◽  
Chromosome 5 ◽  
Mutant Genes ◽  
Male Sterility Gene

Bulked segregant analysis (BSA) assists in map-based cloning of mutant genes. However, a traditional BSA requires many high-density markers for successful linkage analysis which is labor-intensive and time-consuming. In this study, a semi-quantitative DNA analysis program was optimized and combined it with BSA, resulting in a semi-quantitative BSA (sq-BSA). The sq-BSA approach allowed evaluation of the proportions of marker-defined individuals (dominant or recessive marker types) in bulks. The sq-BSA method was used to map a male-sterility (ms) gene, ms2016, in maize. Forty polymorphic markers were screened from one-third of each chromosome (from the head or tail) for mapping. Among these markers, seven were identified as candidate gene-linked markers, of which four markers (bnlg1046, umc1563, umc1171 and umc1722) were located on chromosome 5. Using group validation, ms2016 was anchored on chromosome 5 and was most closely linked to bnlg1046. Furthermore, four new InDel markers located near bnlg1046 were screened to map the preliminary location of ms2016. The ms2016 gene was mapped to an 8.7 Mb interval flanked by the InDel polymorphic markers I5-3 (chr5:14588060) and I5-12 (chr5:23308445). Thus, this improved BSA method (sq-BSA) requires only a small number of molecular markers to quickly localize a target gene, representing a high-efficiency tool for mutant gene mapping. © 2021 Friends Science Publishers

scholarly journals Assessment of a split homing based gene drive for efficient knockout of multiple genes

2019 ◽  
Author(s):  
Nikolay P. Kandul ◽  
Junru Liu ◽  
Anna Buchman ◽  
Valentino M. Gantz ◽  
Ethan Bier ◽  
...  
Keyword(s):  
Target Gene ◽  
Tissue Specificity ◽  
Target Genes ◽  
High Efficiency ◽  
Population Control ◽  
Natural Populations ◽  
Pathogen Transmission ◽  
Gene Drive ◽  
Guide Rnas ◽  
Gene Drives

AbstractHoming based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host-encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate host encoded genes has not been thoroughly explored. To test this approach, here we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of its target gene during super-Mendelian propagation of split-HGD. However, maternal deposition and embryonic expression of Cas9 resulted in the generation of drive resistant alleles which can accumulate and limit the spread of such a drive. Alternative design principles are discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.

scholarly journals Gene activation by a CRISPR-assistant trans enhancer

2019 ◽  
Author(s):  
Xin Hui Xu ◽  
Wei Dai ◽  
Dan Yang Wang ◽  
Jian Wu ◽  
Jin Ke Wang
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
High Efficiency ◽  
Gene Activation ◽  
Biological Research ◽  
Activation Domain ◽  
Activation Efficiency ◽  
New Strategy ◽  
Cmv Enhancer ◽  
Endogenous Genes

AbstractGene activation is essential to the basic biological research and biomedicine. Therefore, various gene activators such as activation domain-ZNF, TALE and CRISPR proteins have been developed for this end, in which the CRISPR protein dead Cas9 (dCas9) is now most widely used. However, the current gene activators are still limited by their inefficient gene activation activity. In this study, we developed a new strategy, CRISPR-assistant trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with a widely used strong enhancer, the CMV enhancer. In this strategy, a trans CMV enhancer DNA was recruited to target gene by dCas9-VP64/sgRNA via annealing between 3’ end of sgRNA and CMV enhancer. The trans enhancer activates gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.

Microcalorimeters for X-Ray Spectroscopy

1988 ◽  
Vol 102 ◽  
pp. 41
Author(s):  
E. Silver ◽  
C. Hailey ◽  
S. Labov ◽  
N. Madden ◽  
D. Landis ◽  
...  
Keyword(s):  
High Resolution ◽  
High Efficiency ◽  
Thermal Response ◽  
Low Noise ◽  
High Resolution Spectroscopy ◽  
Superconducting Transition ◽  
Sapphire Substrates ◽  
Laboratory Plasmas ◽  
Adiabatic Demagnetization ◽  
Theoretical Predictions

The merits of microcalorimetry below 1°K for high resolution spectroscopy has become widely recognized on theoretical grounds. By combining the high efficiency, broadband spectral sensitivity of traditional photoelectric detectors with the high resolution capabilities characteristic of dispersive spectrometers, the microcalorimeter could potentially revolutionize spectroscopic measurements of astrophysical and laboratory plasmas. In actuality, however, the performance of prototype instruments has fallen short of theoretical predictions and practical detectors are still unavailable for use as laboratory and space-based instruments. These issues are currently being addressed by the new collaborative initiative between LLNL, LBL, U.C.I., U.C.B., and U.C.D.. Microcalorimeters of various types are being developed and tested at temperatures of 1.4, 0.3, and 0.1°K. These include monolithic devices made from NTD Germanium and composite configurations using sapphire substrates with temperature sensors fabricated from NTD Germanium, evaporative films of Germanium-Gold alloy, or material with superconducting transition edges. A new approache to low noise pulse counting electronics has been developed that allows the ultimate speed of the device to be determined solely by the detector thermal response and geometry. Our laboratory studies of the thermal and resistive properties of these and other candidate materials should enable us to characterize the pulse shape and subsequently predict the ultimate performance. We are building a compact adiabatic demagnetization refrigerator for conveniently reaching 0.1°K in the laboratory and for use in future satellite-borne missions. A description of this instrument together with results from our most recent experiments will be presented.

Imaging and diffraction modes in Scanning Transmission Electron Microscopy

1986 ◽  
Vol 44 ◽  
pp. 684-687
Author(s):  
J. M. Cowley ◽  
R. Glaisher ◽  
J. A. Lin ◽  
H.-J. Ou
Keyword(s):  
Scanning Transmission Electron Microscopy ◽  
High Efficiency ◽  
Fiber Optic ◽  
Image Intensifier ◽  
Detector System ◽  
Detector Plane ◽  
Secondary Electrons ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Special Detector

Some of the most important applications of STEM depend on the variety of imaging and diffraction made possible by the versatility of the detector system and the serial nature, of the image acquisition. A special detector system, previously described, has been added to our STEM instrument to allow us to take full advantage of this versatility. In this, the diffraction pattern in the detector plane may be formed on either of two phosphor screens, one with P47 (very fast) phosphor and the other with P20 (high efficiency) phosphor. The light from the phosphor is conveyed through a fiber-optic rod to an image intensifier and TV system and may be photographed, recorded on videotape, or stored digitally on a frame store. The P47 screen has a hole through it to allow electrons to enter a Gatan EELS spectrometer. Recently a modified SEM detector has been added so that high resolution (10Å) imaging with secondary electrons may be used in conjunction with other modes.

Influence of MBE Growth Conditions on Dislocation Densities of GaAs/Ge Epilayers Grown on Silicon Substrates

1985 ◽  
Vol 43 ◽  
pp. 364-365
Author(s):  
K.M. Hones ◽  
P. Sheldon ◽  
B.G. Yacobi ◽  
A. Mason
Keyword(s):  
High Efficiency ◽  
Lattice Mismatch ◽  
Low Cost ◽  
Growth Conditions ◽  
Nonradiative Recombination ◽  
Device Structure ◽  
Si Substrates ◽  
Transmission Electron ◽  
Dislocation Densities ◽  
Dislocation Type

There is increasing interest in growing epitaxial GaAs on Si substrates. Such a device structure would allow low-cost substrates to be used for high-efficiency cascade- junction solar cells. However, high-defect densities may result from the large lattice mismatch (∼4%) between the GaAs epilayer and the silicon substrate. These defects can act as nonradiative recombination centers that can degrade the optical and electrical properties of the epitaxially grown GaAs. For this reason, it is important to optimize epilayer growth conditions in order to minimize resulting dislocation densities. The purpose of this paper is to provide an indication of the quality of the epitaxially grown GaAs layers by using transmission electron microscopy (TEM) to examine dislocation type and density as a function of various growth conditions. In this study an intermediate Ge layer was used to avoid nucleation difficulties observed for GaAs growth directly on Si substrates. GaAs/Ge epilayers were grown by molecular beam epitaxy (MBE) on Si substrates in a manner similar to that described previously.

The use of high-resolution FESEM to study solid-state phase transformations and reactions

1994 ◽  
Vol 52 ◽  
pp. 1028-1029
Author(s):  
P. G. Kotula ◽  
D. D. Erickson ◽  
C. B. Carter
Keyword(s):  
Solid State ◽  
High Resolution ◽  
Phase Transformations ◽  
High Efficiency ◽  
Sol Gel ◽  
Quantitative Information ◽  
Solid State Reactions ◽  
Critical Dimensions ◽  
Backscattered Electrons ◽  
Backscattered Electron

High-resolution field-emission-gun scanning electron microscopy (FESEM) has recently emerged as an extremely powerful method for characterizing the micro- or nanostructure of materials. The development of high efficiency backscattered-electron detectors has increased the resolution attainable with backscattered-electrons to almost that attainable with secondary-electrons. This increased resolution allows backscattered-electron imaging to be utilized to study materials once possible only by TEM. In addition to providing quantitative information, such as critical dimensions, SEM is more statistically representative. That is, the amount of material that can be sampled with SEM for a given measurement is many orders of magnitude greater than that with TEM.In the present work, a Hitachi S-900 FESEM (operating at 5kV) equipped with a high-resolution backscattered electron detector, has been used to study the α-Fe2O3 enhanced or seeded solid-state phase transformations of sol-gel alumina and solid-state reactions in the NiO/α-Al2O3 system. In both cases, a thin-film cross-section approach has been developed to facilitate the investigation. Specifically, the FESEM allows transformed- or reaction-layer thicknesses along interfaces that are millimeters in length to be measured with a resolution of better than 10nm.

Interlayer confinement synthesis of Ir nanodots/dual carbon as an electrocatalyst for overall water splitting

2020 ◽  
Author(s):  
Yaru Li ◽  
Yu-Quan Zhu ◽  
Weili Xin ◽  
Song Hong ◽  
Xiaoying Zhao ◽  
...  
Keyword(s):  
Water Splitting ◽  
Noble Metal ◽  
High Efficiency ◽  
Overall Water Splitting ◽  
Highly Dispersed

Rationally designing low-content and high-efficiency noble metal nanodots offers opportunities to enhance electrocatalytic performances for water splitting. However, the preparation of highly dispersed nanodots electrocatalysts remains a challenge. Herein, we...

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