scholarly journals An Improved Method to Knock Out theasdGene ofSalmonella entericaSerovar Pullorum

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Shi-Zhong Geng ◽  
Xin-An Jiao ◽  
Zhi-Ming Pan ◽  
Xiao-Juan Chen ◽  
Xiao-Ming Zhang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Gene Knockout ◽  
Vector System ◽  
Improved Method ◽  
Suicide Plasmid ◽  
Knock Out ◽  
Suicide Vector ◽  
Conventional Methods

Anasd-deleted (Δasd) mutant ofSalmonella entericaserovar Pullorum (SP) was constructed using an improved method of gene knockout by combining theπ-suicide plasmid system with the Red Disruption system. Theasdgene was efficiently knocked out by the recombinant suicide vector, which replaced theasdgene with theCmRgene. Based on the balanced lethal host-vector system, the phenotype of theΔasdmutant was further defined. The improved method was simpler and more effective than previously reported conventional methods.

Insertion-Duplication Mutagenesis inStreptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool

1998 ◽  
Vol 64 (12) ◽  
pp. 4796-4802 ◽  
Author(s):  
Myeong S. Lee ◽  
Chaok Seok ◽  
Donald A. Morrison
Keyword(s):  
Gene Knockout ◽  
Genomic Analysis ◽  
Combinatorial Theory ◽  
Donor Plasmid ◽  
Uniform Size ◽  
Knock Out ◽  
Duplication Events ◽  
Simplifying Assumptions ◽  
Insertion Tool ◽  
Random Insertion

ABSTRACT To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

scholarly journals In silico gene knockout prediction using a hybrid of Bat algorithm and minimization of metabolic adjustment

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mei Yen Man ◽  
Mohd Saberi Mohamad ◽  
Yee Wen Choon ◽  
Mohd Arfian Ismail
Keyword(s):  
Metabolic Engineering ◽  
Gene Knockout ◽  
Lactate Production ◽  
Bat Algorithm ◽  
Host Cells ◽  
Knock Out ◽  
Genetic Manipulations ◽  
Microbial Strains ◽  
Metabolic Adjustment ◽  
Minimization Of Metabolic Adjustment

Abstract Microorganisms commonly produce many high-demand industrial products like fuels, food, vitamins, and other chemicals. Microbial strains are the strains of microorganisms, which can be optimized to improve their technological properties through metabolic engineering. Metabolic engineering is the process of overcoming cellular regulation in order to achieve a desired product or to generate a new product that the host cells do not usually need to produce. The prediction of genetic manipulations such as gene knockout is part of metabolic engineering. Gene knockout can be used to optimize the microbial strains, such as to maximize the production rate of chemicals of interest. Metabolic and genetic engineering is important in producing the chemicals of interest as, without them, the product yields of many microorganisms are normally low. As a result, the aim of this paper is to propose a combination of the Bat algorithm and the minimization of metabolic adjustment (BATMOMA) to predict which genes to knock out in order to increase the succinate and lactate production rates in Escherichia coli (E. coli).

scholarly journals Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

2019 ◽  
Vol 294 (25) ◽  
pp. 9734-9745 ◽  
Author(s):  
Chong Wang ◽  
Sizun Jiang ◽  
Liangru Ke ◽  
Luyao Zhang ◽  
Difei Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Advanced Disease ◽  
Gene Knockout ◽  
Histone Acetyl Transferase ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Cellular Factors ◽  
New Treatments ◽  
Specific Symptoms

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

scholarly journals CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans

2021 ◽  
Vol 22 (21) ◽  
pp. 11423
Author(s):  
Chunxin Liu ◽  
Yiyao Zhang ◽  
Yinxiao Tan ◽  
Tingting Zhao ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Late Blight ◽  
Phytophthora Infestans ◽  
Gene Knockout ◽  
Disease Index ◽  
Oxygen Species ◽  
Wild Type ◽  
Pr Genes ◽  
Knock Out ◽  
Pathogenesis Related

Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.

scholarly journals Transduction-Mediated Transfer of Unmarked Deletion and Point Mutations through Use of Counterselectable Suicide Vectors

2002 ◽  
Vol 184 (1) ◽  
pp. 307-312 ◽  
Author(s):  
Ho Young Kang ◽  
Charles M. Dozois ◽  
Steven A. Tinge ◽  
Tae Ho Lee ◽  
Roy Curtiss
Keyword(s):  
Salmonella Enterica ◽  
Tandem Duplication ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Allelic Exchange ◽  
Efficient Strategy ◽  
Suicide Vector ◽  
Serovar Typhimurium ◽  
Generalized Transduction ◽  
Mutant Chromosome

ABSTRACT A challenge in strain construction is that unmarked deletion and nucleotide substitution alleles generally do not confer selectable phenotypes. We describe here a rapid and efficient strategy for transferring such alleles via generalized transduction. The desired allele is first constructed and introduced into the chromosome by conventional allelic-exchange methods. The suicide vector containing the same allele is then integrated into the mutant chromosome, generating a tandem duplication homozygous for that allele. The resulting strain is used as a donor for transductional crosses, and selection is made for a marker carried by the integrated suicide vector. Segregation of the tandem duplication results in haploid individuals, each of which carries the desired allele. To demonstrate this mutagenesis strategy, we used bacteriophage P22Hint for generalized transduction-mediated introduction of unmarked mutations to Salmonella enterica serovar Typhimurium. This method is applicable to any species for which generalized transduction is established.

scholarly journals Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1

2014 ◽  
Vol 63 (6) ◽  
pp. 788-795 ◽  
Author(s):  
Inke Wallrodt ◽  
Lotte Jelsbak ◽  
Line E. Thomsen ◽  
Lena Brix ◽  
Sébastien Lemire ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Single Gene ◽  
Systemic Infection ◽  
Minor Effect ◽  
Membrane Stress ◽  
Knock Out ◽  
Serovar Typhimurium ◽  
A Minor

The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA–D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1+ mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA–D set of genes caused attenuation in both NRAMP1+ and NRAMP1− mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1+ mice, and deletion of pspD had a minor effect in NRAMP1− mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA.

scholarly journals Unsupervised Spike Detection and Sorting with Wavelets and Superparamagnetic Clustering

2004 ◽  
Vol 16 (8) ◽  
pp. 1661-1687 ◽  
Author(s):  
R. Quian Quiroga ◽  
Z. Nadasdy ◽  
Y. Ben-Shaul
Keyword(s):  
Simulated Data ◽  
New Method ◽  
Data Sets ◽  
Improved Method ◽  
Spike Detection ◽  
Gaussian Distributions ◽  
Conventional Methods ◽  
Simulated Data Sets

This study introduces a new method for detecting and sorting spikes from multiunit recordings. The method combines the wave let transform, which localizes distinctive spike features, with super paramagnetic clustering, which allows automatic classification of the data without assumptions such as low variance or gaussian distributions. Moreover, an improved method for setting amplitude thresholds for spike detection is proposed. We describe several criteria for implementation that render the algorithm unsupervised and fast. The algorithm is compared to other conventional methods using several simulated data sets whose characteristics closely resemble those of in vivo recordings. For these data sets, we found that the proposed algorithm outperformed conventional methods.

scholarly journals The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

2021 ◽  
Vol 22 (21) ◽  
pp. 11389
Author(s):  
Sang-Tae Kim ◽  
Minkyung Choi ◽  
Su-Ji Bae ◽  
Jin-Soo Kim
Keyword(s):  
Salt Stress ◽  
Target Genes ◽  
Gene Knockout ◽  
Salt Resistance ◽  
Knock Out ◽  
Chlorophyll Contents ◽  
Guide Rnas ◽  
Osmotic Tolerance ◽  
Multiple Genes ◽  
Acclimation Period

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

scholarly journals Noninvasive sleep monitoring in large-scale screening of knock-out mice reveals novel sleep-related genes

2019 ◽  
Author(s):  
Shreyas S. Joshi ◽  
Mansi Sethi ◽  
Martin Striz ◽  
Neil Cole ◽  
James M. Denegre ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Large Scale ◽  
Gene Knockout ◽  
Single Gene ◽  
Mammalian Species ◽  
Sleep Behavior ◽  
Knock Out ◽  
Circadian Phase ◽  
Gene Knockout Mice ◽  
Normal Sleep

AbstractSleep is a critical process that is well-conserved across mammalian species, and perhaps most animals, yet its functions and underlying mechanisms remain poorly understood. Identification of genes and pathways that can influence sleep may shed new light on these functions. Genomic screens enable the detection of previously unsuspected molecular processes that influence sleep. In this study, we report results from a large-scale phenotyping study of sleep-wake parameters for a population of single-gene knockout mice. Sleep-wake parameters were measured using a high throughput, non-invasive piezoelectric system called PiezoSleep. Knockout mice generated on a C57BL6/N (B6N) background were monitored for sleep and wake parameters for five days. By analyzing data from over 6000 mice representing 343 single gene knockout lines, we identified 122 genes influencing traits like sleep duration and bout length that have not been previously implicated in sleep, including those that affect sleep only during a specific circadian phase. PiezoSleep also allows assessment of breath rates during sleep and this was integrated as a supplemental tool in identifying aberrant physiology in these knockout lines. Sex differences were evident in both normal and altered sleep behavior. Through a combination of genetic and phenotypic associations, and known QTLs for sleep, we propose a set of candidate genes playing specific roles in sleep. The high “hit rate” demonstrates that many genes can alter normal sleep behaviors through a variety of mechanisms. Further investigation of these genes may provide insight into the pathways regulating sleep, functional aspects of sleep, or indirect potentially pathological processes that alter normal sleep.

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