scholarly journals Use of Large-Scale Expression Cloning Screens in the Xenopus laevis Tadpole to Identify Gene Function

2000 ◽  
Vol 228 (2) ◽  
pp. 197-210 ◽  
Author(s):  
Timothy C Grammer ◽  
Karen J Liu ◽  
Francesca V Mariani ◽  
Richard M Harland
Keyword(s):  
Xenopus Laevis ◽  
Gene Function ◽  
Large Scale ◽  
Expression Cloning ◽  
Xenopus Laevis Tadpole

scholarly journals A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michelle Spoto ◽  
Changhui Guan ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Model Organisms ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Wide Range ◽  
User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

scholarly journals Large-scale gene function analysis with the PANTHER classification system

2013 ◽  
Vol 8 (8) ◽  
pp. 1551-1566 ◽  
Author(s):  
Huaiyu Mi ◽  
Anushya Muruganujan ◽  
John T Casagrande ◽  
Paul D Thomas
Keyword(s):  
Gene Function ◽  
Classification System ◽  
Large Scale ◽  
Function Analysis ◽  
Gene Function Analysis

Large-scale gene function analysis in Candida albicans

2004 ◽  
Vol 12 (4) ◽  
pp. 157-161 ◽  
Author(s):  
Vincent M. Bruno ◽  
Aaron P. Mitchell
Keyword(s):  
Candida Albicans ◽  
Gene Function ◽  
Large Scale ◽  
Function Analysis ◽  
Gene Function Analysis

scholarly journals Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

1987 ◽  
Vol 104 (4) ◽  
pp. 841-847 ◽  
Author(s):  
BK Kay ◽  
AJ Shah ◽  
WE Halstead
Keyword(s):  
Xenopus Laevis ◽  
Fusion Protein ◽  
Binding Protein ◽  
Expression Library ◽  
Simple Method ◽  
High Affinity ◽  
Tadpole Tail ◽  
Developing Muscle ◽  
Beta Galactosidase ◽  
Xenopus Laevis Tadpole

A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta-parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.

Large scale isolation of nuclei and nucleoli from vitellogenic oocytes of Xenopus laevis

Chromosoma ◽  
1978 ◽  
Vol 66 (4) ◽  
pp. 299-308 ◽  
Author(s):  
Franco Scalenghe ◽  
Marino Buscaglia ◽  
Christine Steinheil ◽  
Marco Crippa
Keyword(s):  
Xenopus Laevis ◽  
Large Scale

scholarly journals A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

2005 ◽  
Vol 18 (8) ◽  
pp. 755-761 ◽  
Author(s):  
D. Douchkov ◽  
D. Nowara ◽  
U. Zierold ◽  
P. Schweizer
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Gene Function ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Powdery Mildew Fungus ◽  
Nonspecific Resistance ◽  
Knock Down ◽  
Highly Efficient

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE proteinin three types of durable, race-nonspecific resistance was observed.

Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies

1987 ◽  
Vol 122 (1) ◽  
pp. 90-100 ◽  
Author(s):  
Shin Takagi ◽  
Toshiaki Tsuji ◽  
Takashi Amagai ◽  
Tetsuro Takamatsu ◽  
Hajime Fujisawa
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Xenopus Laevis ◽  
Specific Cell ◽  
Specific Cell Surface ◽  
Xenopus Laevis Tadpole
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