scholarly journals An Efficient Workflow for Screening and Stabilizing CRISPR/Cas9-Mediated Mutant Lines in Bombyx mori

2020 ◽  
Vol 4 (1) ◽  
pp. 4
Author(s):  
Daniel Brady ◽  
Alessio Saviane ◽  
Silvia Cappellozza ◽  
Federica Sandrelli
Keyword(s):  
Bombyx Mori ◽  
Target Genes ◽  
Economic Value ◽  
Model Organism ◽  
Amplification Refractory Mutation System ◽  
Induced Mutations ◽  
Mutation System ◽  
Stable Mutant ◽  
Sequential Combination ◽  
Mutant Lines

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects.

Predictive role of single nucleotide polymorphism (rs11614913) in the development of breast cancer in Pakistani population

2020 ◽  
Vol 17 (3) ◽  
pp. 213-227
Author(s):  
Mushtaq Ahmad ◽  
Aftab Ali Shah
Keyword(s):  
Breast Cancer ◽  
Single Nucleotide Polymorphism ◽  
Target Genes ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Significant Difference ◽  
Mutation System ◽  
Predictive Role

Aim: miRNAs play an important role in breast cancer (BC). Variations in miRNAs influence their maturation, expression and consequently regulation of their target genes. Materials & methods: In this study, single nucleotide polymorphism rs11614913 was genotyped in BC patients (n = 300) and 230 controls by employing tetra primer amplification refractory mutation system PCR and Sanger sequencing (Macrogen Korea). Results: A significant difference was observed in the genotypes through co-dominant ( χ2.#x00A0;= 42.03; p < 0.0001), additive (odds ratio [OR] = 0.6441 [0.4887–0.8490, 95% confidence interval]; p < 0.0019), dominant (OR = 0.3996 [0.2809–0.5686], p < 0.0001) and recessive (OR = 0.2993 [0.1220–0.7347], p < 0.009) statistical models showed decreased risk association of C allele with BC. Conclusion: Females having CT genotype are at higher risk of BC as compared with those having CC genotype.

scholarly journals Deacetylation of HSC70-4 Promotes Bombyx mori Nucleopolyhedrovirus Proliferation via Proteasome-Mediated Nuclear Import

2021 ◽  
Vol 12 ◽  
Author(s):  
Fuxiang Mao ◽  
Xi Chen ◽  
Jonas Ngowo ◽  
Yajie Zhu ◽  
Jihai Lei ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Economic Value ◽  
Model Organism ◽  
Ubiquitin Proteasome System ◽  
Nuclear Accumulation ◽  
Genome Replication ◽  
Nuclear Entry ◽  
Interacting Protein ◽  
Viral Pathogen ◽  
Biological Studies

Silkworm (Bombyx mori) is a model organism with great agricultural economic value that plays a crucial role in biological studies. B. mori nucleopolyhedrovirus (BmNPV) is a major viral pathogen found in silkworms, which leads to huge silk loss annually. In a recent lysine acetylome of silkworm infected with BmNPV, we focused on the heat shock cognate protein 70-4 (HSC70-4) lysine acetylation change due to the consequent nuclear accumulation and viral structure assembly. In this study, the genome replication, proliferation, and production of budded viruses (BVs) were arrested by HSP/HSC70 inhibitor treatment. However, HSC70-4 overexpression enhanced BmNPV reproduction. Furthermore, site-direct mutagenesis for acetylated mimic (K/Q) or deacetylated mimic (K/R) mutants of HSC70-4 demonstrated that lysine 77 (K77) deacetylation promotes HSC70-4 stability, viral DNA duplication, and HSC70-4 nuclear entry upon BmNPV challenge, and the nuclear propulsion of HSC70-4 after viral stimulus might be dependent on the interaction with the carboxyl terminus of HSC70-interacting protein (CHIP, an E3 ubiquitin ligase), followed by ubiquitin-proteasome system assistance. In this study, single lysine 77 deacetylation of HSC70-4 was deemed a part of the locomotive pathway for facilitating BmNPV proliferation and provided novel insights into the antiviral strategic development.

scholarly journals Homology Directed Repair in Zebrafish: Witchcraft and Wizardry?

2020 ◽  
Author(s):  
Kendal Prill ◽  
John F Dawson
Keyword(s):  
Protein Function ◽  
Target Genes ◽  
Chemical Mutagenesis ◽  
Model Organisms ◽  
Zinc Finger Nucleases ◽  
Success Rates ◽  
Homology Directed Repair ◽  
Stable Mutant ◽  
Genetic Techniques ◽  
Mutant Lines

Introducing desired mutations into the genome of model organisms is a priority for all research focusing on protein function and disease modelling. The need to create stable mutant lines has resulted in the rapid advancement of genetic techniques over the last few decades from chemical mutagenesis and zinc finger nucleases to CRISPR and Homology Directed Repair. However, achieving consistently high success rates for direct mutagenesis in zebrafish remains one of the most sought after techniques in the field. Several genes have been modified using HDR in zebrafish but published success rates range widely suggesting that an optimal protocol is required. In this review, we compare target genes, techniques and protocols from 37 genes that were successfully modified in zebrafish using HDR to find the statistically best variables for efficient HDR rates.

scholarly journals Amplification Refractory Mutation System (ARMS)-PCR for Waxy Sorghum Authentication with Single-Nucleotide Resolution

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2218
Author(s):  
Xiaoying Zhu ◽  
Minghua Wu ◽  
Ruijie Deng ◽  
Mohammad Rizwan Khan ◽  
Sha Deng ◽  
...  
Keyword(s):  
Economic Value ◽  
High Sensitivity ◽  
Amplification Refractory Mutation System ◽  
Brewing Industry ◽  
Food Authentication ◽  
Single Nucleotide ◽  
Mutation System ◽  
Pcr Method ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Waxy sorghum has greater economic value than wild sorghum in relation to their use in food processing and the brewing industry. Thus, the authentication of the waxy sorghum species is an important issue. Herein, a rapid and sensitive Authentication Amplification Refractory Mutation System-PCR (aARMS-PCR) method was employed to identify sorghum species via its ability to resolve single-nucleotide in genes. As a proof of concept, we chose a species of waxy sorghum containing the wxc mutation which is abundantly used in liquor brewing. The aARMS-PCR can distinguish non-wxc sorghum from wxc sorghum to guarantee identification of specific waxy sorghum species. It allowed to detect as low as 1% non-wxc sorghum in sorghum mixtures, which ar one of the most sensitive tools for food authentication. Due to its ability for resolving genes with single-nucleotide resolution and high sensitivity, aARMS-PCR may have wider applicability in monitoring food adulteration, offering a rapid food authenticity verification in the control of adulteration.

scholarly journals Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?

2020 ◽  
Vol 7 ◽  
Author(s):  
Kendal Prill ◽  
John F. Dawson
Keyword(s):  
Protein Function ◽  
Target Genes ◽  
Disease Modeling ◽  
Chemical Mutagenesis ◽  
Model Organisms ◽  
Zinc Finger Nucleases ◽  
Success Rates ◽  
Homology Directed Repair ◽  
Stable Mutant ◽  
Mutant Lines

Introducing desired mutations into the genome of model organisms is a priority for all research focusing on protein function and disease modeling. The need to create stable mutant lines has resulted in the rapid advancement of genetic techniques over the last few decades from chemical mutagenesis and zinc finger nucleases to clustered regularly interspaced short palindromic repeats (CRISPR) and homology-directed repair (HDR). However, achieving consistently high success rates for direct mutagenesis in zebrafish remains one of the most sought-after techniques in the field. Several genes have been modified using HDR in zebrafish, but published success rates range widely, suggesting that an optimal protocol is required. In this review, we compare target genes, techniques, and protocols from 50 genes that were successfully modified in zebrafish using HDR to find the statistically best variables for efficient HDR rates.

Genetic Variation in TNF-α, its Relation with Inflammatory Biomarkers and Susceptibility to Rheumatoid Arthritis

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Khadiga Ahmed Ismail
Keyword(s):  
Rheumatoid Arthritis ◽  
Serum Level ◽  
Functional Disability ◽  
Serum Levels ◽  
Amplification Refractory Mutation System ◽  
Reactive Protein ◽  
Tnf Α ◽  
Mutation System ◽  
Potential Factor ◽  
Factor Α

Background: Tumor necrosis Factor-α (TNF-α) is encoded and controlled by TNF-α gene, which is involved in rheumatoid arthritis (RA) susceptibility. This research aimed to identify genetic variations of TNF-α (G308A) and to establish its association with inflammatory markers in Rheumatoid Arthritis predisposition. Methods: In the present study, fifty RA patients and fifty volunteers were involved and evaluated for the C-reactive protein, rheumatoid factor, and TNF-α were estimated by ELISA, Erythrocyte Sedimentation Rate (ESR) by Wintergreen method and for TNF-α-308 G>A polymorphism by polymerase chain reaction with amplification refractory mutation system (PCR-ARMS). Results: The CRP, RF, ESR and TNF-α were significantly elevated in RA patients relative to controls. The serum level TNF-α was also significantly elevated in female patients and in patients ≥50 years. Analysis of TNF-308 gene polymorphism revealed that GG genotypes were more prevalent in RA patients than in the healthy individuals and that GG genotype may be a potential factor to RA. The G allele was more common in RA than in the control. Elevated TNF-α serum levels were significantly associated the GG genotype and functional disability in RA patients. Conclusion: TNF-α promoter 308polymorphism GG genotype may be considered as a risk factor for RA and the TNF-α serum level was significantly related to the functional disability in the disease.

scholarly journals Mutant Lines of Spring Wheat with Increased Iron, Zinc, and Micronutrients in Grains and Enhanced Bioavailability for Human Health

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Saule Kenzhebayeva ◽  
Alfia Abekova ◽  
Saule Atabayeva ◽  
Gulzira Yernazarova ◽  
Nargul Omirbekova ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Protein Content ◽  
Spring Wheat ◽  
Grain Protein Content ◽  
Grain Protein ◽  
Cereal Products ◽  
Molar Ratios ◽  
Stable Mutant ◽  
Micronutrient Malnutrition ◽  
Mutant Lines

Deficiency of metals, primarily Fe and Zn, affects over half of the world’s population. Human diets dominated by cereal products cause micronutrient malnutrition, which is common in many developing countries where populations depend heavily on staple grain crops such as wheat, maize, and rice. Biofortification is one of the most effective approaches to alleviate malnutrition. Genetically stable mutant spring wheat lines (M7 generation) produced via 100 or 200 Gy gamma treatments to broaden genetic variation for grain nutrients were analyzed for nutritionally important minerals (Ca, Fe, and Zn), their bioavailability, and grain protein content (GPC). Variation was 172.3–883.0 mg/kg for Ca, 40.9–89.0 mg/kg for Fe, and 22.2–89.6 mg/kg for Zn. In mutant lines, among the investigated minerals, the highest increases in concentrations were observed in Fe, Zn, and Ca when compared to the parental cultivar Zhenis. Some mutant lines, mostly in the 100 Gy-derived germplasm, had more than two-fold higher Fe, Zn, and Ca concentrations, lower phytic acid concentration (1.4–2.1-fold), and 6.5–7% higher grain protein content compared to the parent. Variation was detected for the molar ratios of Ca:Phy, Phy:Fe, and Phy:Zn (1.27–10.41, 1.40–5.32, and 1.78–11.78, respectively). The results of this study show how genetic variation generated through radiation can be useful to achieve nutrient biofortification of crops to overcome human malnutrition.

scholarly journals Evaluation of AXIN1 and AXIN2 as targets of tankyrase inhibition in hepatocellular carcinoma cell lines

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenhui Wang ◽  
Pengyu Liu ◽  
Marla Lavrijsen ◽  
Shan Li ◽  
Ruyi Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Therapeutic Option ◽  
Carcinoma Cell Lines ◽  
Hepatocellular Carcinomas ◽  
Enhanced Expression ◽  
Mutant Lines ◽  
Substantial Interest ◽  
Support Tumor Growth ◽  
Made In

AbstractAXIN1 mutations are observed in 8–10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling.

scholarly journals Applicability of Honey on Silkworms (Bombyx mori) and Quality Improvement of Its Biomaterials

2021 ◽  
Vol 11 (10) ◽  
pp. 4613
Author(s):  
Gabriela-Maria Baci ◽  
Alexandra-Antonia Cucu ◽  
Adela Ramona Moise ◽  
Daniel Severus Dezmirean
Keyword(s):  
Quality Improvement ◽  
Bombyx Mori ◽  
Human Health ◽  
Recombinant Proteins ◽  
Therapeutic Agent ◽  
Life Sciences ◽  
Model Organism ◽  
Medical Field ◽  
Wide Range ◽  
Ancient Times

Since ancient times, honey has been considered one of the most illustrious and esteemed natural products. Honey plays two key roles; specifically, it is an appreciated nutritional product, and also exhibits a wide range of beneficial properties for human health as a therapeutic agent. Furthermore, it has been shown that honey has valuable effects on the biological and physiological features of mulberry silkworms (Bombyx mori). Bombyx mori exhibits importance not only for the economy, but it also serves as an important biotechnological bioreactor for the production of recombinant proteins that have a great impact in the medical field and beyond. It also represents an important model organism for life sciences. In view of the fact that silk fibroin serves as a natural biopolymer that displays high biocompatibility with human organisms and due to honey’s various and remarkable properties for human health, the two elements are currently used together in order to develop ideal biomaterials for a wide range of purposes. In this review, by discussing the applicability of honey on Bombyx mori and beyond, the importance of honey for life sciences and related fields is spotlighted.

Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in the Shenzhen Population

2021 ◽  
pp. 1-7
Author(s):  
Jian Gao ◽  
Sheng Lin ◽  
Shiguo Chen ◽  
Qunyan Wu ◽  
Kaifeng Zheng ◽  
...  
Keyword(s):  
G6pd Deficiency ◽  
Amplification Refractory Mutation System ◽  
Gene Variants ◽  
Coding Region ◽  
G6pd Gene ◽  
Mutation System ◽  
Hotspot Mutations ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Generation Sequencing

<b><i>Background:</i></b> Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. This study aimed to characterize the G6PD gene variant distribution in Shenzhen of Guangdong province. <b><i>Methods:</i></b> A total of 33,562 individuals were selected at the hospital for retrospective analysis, of which 1,213 cases with enzymatic activity-confirmed G6PD deficiency were screened for G6PD gene variants. Amplification refractory mutation system PCR was first used to screen the 6 dominant mutants in the Chinese population (c.1376G&#x3e;T, c.1388G&#x3e;A, c.95A&#x3e;G, c.1024C&#x3e;T, c.392G&#x3e;T, and c.871G&#x3e;A). If the 6 hotspot variants were not found, next-generation sequencing was then performed. Finally, Sanger sequencing was used to verify all the mutations. <b><i>Results:</i></b> The incidence of G6PD deficiency in this study was 3.54%. A total of 26 kinds of mutants were found in the coding region, except for c.-8-624T&#x3e;C, which was in the noncoding region. c.1376G&#x3e;T and c.1388G&#x3e;A, both located in exon 12, were the top 2 mutants, accounting for 68.43% of all individuals. The 6 hotspot mutations had a cumulative proportion of 94.02%. <b><i>Conclusions:</i></b> This study provided detailed characteristics of G6PD gene variants in Shenzhen, and the results would be valuable to enrich the knowledge of G6PD deficiency.

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