scholarly journals A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
François Kroll ◽  
Gareth T Powell ◽  
Marcus Ghosh ◽  
Gaia Gestri ◽  
Paride Antinucci ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Rapid Screening ◽  
Genetic Screens ◽  
Behavioural Phenotype ◽  
Larval Zebrafish ◽  
Complex Phenotypes ◽  
Human Genes ◽  
Behavioural Phenotypes ◽  
Mutant Phenotypes ◽  
Genetic Contributions

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

scholarly journals A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

2020 ◽  
Author(s):  
François Kroll ◽  
Gareth T Powell ◽  
Marcus Ghosh ◽  
Paride Antinucci ◽  
Timothy J Hearn ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Rapid Screening ◽  
Genetic Screens ◽  
Behavioural Phenotype ◽  
Larval Zebrafish ◽  
Complex Phenotypes ◽  
Human Genes ◽  
Behavioural Phenotypes ◽  
Mutant Phenotypes ◽  
Genetic Contributions

ABSTRACTHundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Rapid screening of glomerular slit diaphragm integrity in larval zebrafish

2007 ◽  
Vol 293 (5) ◽  
pp. F1746-F1750 ◽  
Author(s):  
Dirk M. Hentschel ◽  
Michael Mengel ◽  
Lisa Boehme ◽  
Fabian Liebsch ◽  
Caroline Albertin ◽  
...  
Keyword(s):  
Gene Array ◽  
Slit Diaphragm ◽  
Rapid Screening ◽  
Functional Screening ◽  
Screening Assay ◽  
Puromycin Aminonucleoside ◽  
Zebrafish Model ◽  
Larval Zebrafish ◽  
Large Numbers ◽  
Pharmacological Screening

Gene array-type experiments have identified large numbers of genes thought to be important for the integrity of the glomerular slit diaphragm. Confirmation of individual proteins has been limited by the expenses and time involved in generating transgenic or knockout mice for each candidate. We present a functional screening assay based on the clearance of a 70-kDa fluorescent dextran in another vertebrate system that is rapid and low in cost. In the pronephric glomerulus of larval zebrafish, we have demonstrated quantifiable loss of slit diaphragm integrity in a zebrafish model of puromycin aminonucleoside (PA) toxicity. In addition, after knockdown of CD2-associated protein (CD2AP) and podocin, two well-characterized genetic contributors to podocyte differentiation in mammals, we observed glomerular loss of serum macromolecules similar to that seen in mammalian kidneys with inborn mutations in these genes. Increased filtration of 70-kDa FITC-labeled dextran correlates with effacement of podocyte foot processes in ultrastructural analysis. These findings document the value of the zebrafish model in genomics and pharmacological screening applications.

scholarly journals Ultra-rare variants drive substantial cis-heritability of human gene expression

2017 ◽  
Author(s):  
Ryan D. Hernandez ◽  
Lawrence H. Uricchio ◽  
Kevin Hartman ◽  
Chun Ye ◽  
Andrew Dahl ◽  
...  
Keyword(s):  
Complex Disease ◽  
Rare Variants ◽  
Human Gene ◽  
Purifying Selection ◽  
Sequencing Data ◽  
Inference Procedure ◽  
Mendelian Diseases ◽  
Complex Phenotypes ◽  
Regulatory Architecture ◽  
Human Genes

ABSTRACTThe vast majority of human mutations have minor allele frequencies (MAF) under 1%, with the plurality observed only once (i.e., “singletons”). While Mendelian diseases are predominantly caused by rare alleles, their cumulative contribution to complex phenotypes remains largely unknown. We develop and rigorously validate an approach to jointly estimate the contribution of all alleles, including singletons, to phenotypic variation. We apply our approach to transcriptional regulation, an intermediate between genetic variation and complex disease. Using whole genome DNA and lymphoblastoid cell line RNA sequencing data from 360 European individuals, we conservatively estimate that singletons contribute ~25% of cis-heritability across genes (dwarfing the contributions of other frequencies). Strikingly, the majority (~76%) of singleton heritability derives from ultra-rare variants absent from thousands of additional samples. We develop a novel inference procedure to demonstrate that our results are consistent with rampant purifying selection shaping the regulatory architecture of most human genes.

scholarly journals MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H Thompson ◽  
...  
Keyword(s):  
Axial Skeleton ◽  
Thyroid Stimulating Hormone ◽  
Organ System ◽  
Genetic Screens ◽  
Spatially Distributed ◽  
Functional Understanding ◽  
Increase Productivity ◽  
Brittle Bone Disease ◽  
Mutant Phenotypes ◽  
Deep Phenotyping

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

Rapid Electrochemical Screening of Phenylketonuria Maker Depending on Dehydrogenase Attached to the Pt-Doped Reduced Graphene Oxide Nanocomposites

2021 ◽  
Vol 17 (5) ◽  
pp. 921-931
Author(s):  
Yang Ming ◽  
Yan Yu ◽  
Cheng-Li Yang ◽  
Xin-Mian Chen ◽  
Ru-Xia Han ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Reduced Graphene Oxide ◽  
Limit Of Detection ◽  
Neurological Diseases ◽  
Point Of Care ◽  
Great Sensitivity ◽  
Rapid Screening ◽  
Common Disease ◽  
Detection Range ◽  
Reduced Graphene

Phenylketonuria (PKU) is a common disease associated with amino acid metabolism, and usually occurs in newborns. It can cause serious neurological diseases and even death. However, owing to inadequate-effective treatment, it can only be slowed by a low-phenylalanine (Phe) diet. In addition, PKU screening is essential for newborns in many countries. Therefore, rapid screening is crucial for preventing damage and meeting the large sample diagnosis demand. For confirmed patients, a convenient method to monitor their regular Phe levels is required. However, current clinical methods do not meet the rapid screening and convenient monitoring requirements. Herein, a rapid and facile electrochemical device based on platinum-doped reduced graphene oxide nanocomposites was developed to detect PKU biomarker-Phe. The results demonstrated that the developed electrode has great sensitivity, selectivity, and stability. The detection range was 0.0001 mM to 6 mM with a limit of detection of 0.01 μM. Therefore, this work offers a simple and rapid method for point-of-care PKU screening and daily monitoring.

scholarly journals The adaptive shaping of social behavioural phenotypes during adolescence

2018 ◽  
Vol 14 (11) ◽  
pp. 20180536 ◽  
Author(s):  
Norbert Sachser ◽  
Michael B. Hennessy ◽  
Sylvia Kaiser
Keyword(s):  
Neural Plasticity ◽  
Social Behaviour ◽  
Behavioural Change ◽  
Maternal Behaviour ◽  
Behavioural Plasticity ◽  
Behavioural Phenotype ◽  
Shaping Process ◽  
Behavioural Phenotypes ◽  
Underlying Mechanisms ◽  
High Degree

Developmental behavioural plasticity is a process by which organisms can alter development of their behavioural phenotype to be better adapted to the environment encountered later in life. This ‘shaping’ process depends on the presence of reliable cues by which predictions can be made. It is now established that cues detected by the mother can be used (primarily via hormones prenatally and maternal behaviour in the early postnatal stage) to shape the behavioural phenotype of her offspring. However, it is becoming increasingly clear that adolescence is another period in which conditions are well-suited for such shaping to occur. We review here how mammalian social behaviour may be shaped in adolescence. We identify limited extant examples, briefly discuss underlying mechanisms, and provide evidence that observed changes are indeed adaptive. We contend that while plasticity diminishes with age, the shaping of the behavioural phenotype in adolescence offers several advantages, including that adolescence is closer to the onset of mating than are earlier phases of life; that unlike earlier phases, information is obtained directly from the environment rather than mediated by the mother; and unlike later in adulthood, there is substantial underlying neural plasticity associated with development to support behavioural change. We also consider conditions that favour the occurrence of social behaviour plasticity during adolescence, including a high degree of sociality and a prolonged developmental period and the implication of these conditions for the occurrence of sex differences in the shaping process.

scholarly journals Factors Associated with Mutations: Their Matching Rates to Cardiovascular and Neurological Diseases

2021 ◽  
Vol 22 (10) ◽  
pp. 5057
Author(s):  
Hannah B. Lucas ◽  
Ian McKnight ◽  
Regan Raines ◽  
Abdullah Hijazi ◽  
Christoph Hart ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Nucleotide Polymorphisms ◽  
Factors Associated ◽  
Genome Data ◽  
Rare Mutations ◽  
Human Genes ◽  
A Genome ◽  
Two Factors ◽  
Vascular Stiffening ◽  
Monogenic Hypertension

Monogenic hypertension is rare and caused by genetic mutations, but whether factors associated with mutations are disease-specific remains uncertain. Given two factors associated with high mutation rates, we tested how many previously known genes match with (i) proximity to telomeres or (ii) high adenine and thymine content in cardiovascular diseases (CVDs) related to vascular stiffening. We extracted genomic information using a genome data viewer. In human chromosomes, 64 of 79 genetic loci involving >25 rare mutations and single nucleotide polymorphisms satisfied (i) or (ii), resulting in an 81% matching rate. However, this high matching rate was no longer observed as we checked the two factors in genes associated with essential hypertension (EH), thoracic aortic aneurysm (TAA), and congenital heart disease (CHD), resulting in matching rates of 53%, 70%, and 75%, respectively. A matching of telomere proximity or high adenine and thymine content projects the list of loci involving rare mutations of monogenic hypertension better than those of other CVDs, likely due to adoption of rigorous criteria for true-positive signals. Our data suggest that the factor–disease matching rate is an accurate tool that can explain deleterious mutations of monogenic hypertension at a >80% match—unlike the relatively lower matching rates found in human genes of EH, TAA, CHD, and familial Parkinson’s disease.

scholarly journals THE INTERACTION OF miR-4258, miR-3960, miR-211-3p AND miR-3155b WITH mRNAs GENES OF NON-POLYGLUTAMINE TRINUCLEOTIDE DISORDERS

2020 ◽  
Vol 1 (337) ◽  
pp. 25-32 ◽  
Author(s):  
Belkozhayev A.M. ◽  
Niyazova R.Ye.
Keyword(s):  
Binding Energy ◽  
Binding Sites ◽  
Trinucleotide Repeat ◽  
Neurological Diseases ◽  
Repeat Expansion ◽  
Binding Characteristics ◽  
Human Genes ◽  
Free Binding Energy ◽  
Nucleotide Repeats ◽  
Cug Repeats

Trinucleotide repeat expansion disorders constitute a group of dominantly inherited neurological diseases that are incurable and ultimately fatal. In the present work, miRNA binding sites were predicted by the MirTarget program. It was given characteristics of miRNAs binding sites in 5' and 3' UTR mRNAs genes of non-polyglutamine trinucleotide disorders with CGG, GCC, CUG repeats. Binding sites of 2567 miRNAs with mRNAs of 17494 human genes were determined. 206 genes with nucleotide repeats, mRNAs of which are bind with miRNA in the 5'UTR and 3'UTR, were observed. From thus, 2668 miRNAs binding sites are located in the 5'UTR, 3853 – in the 3'UTR with ΔG/ΔGm values equal to 85 % and more. It was found that 34 gene’s mRNA having trinucleotide (CGG\GCC\CUG) repeats were targets for miR-4258, miR-3960 miR-211-3p and miR-3155b. miR-4258 binds to mRNA of ADARB1, C11orf87 and CBFB genes with free binding energy - 93 kJ/mole and ΔG/ΔGm 91%, to mRNA of ARHGEF7, BCR, BRSK2 and C9orf91 genes with free binding energy - 91 kJ/mole and ΔG/ΔGm 89%. miR-3960 binds in GCC repeats to mRNA of ABCC1 and BLMH genes with free binding energy - 116 kJ/mole. miR-211-3p and miR-3155b interact with mRNA of ACACA and ANKRD13D genes in 5’-3’untranslated regions. Studying binding characteristics of miRNA and genes will help identify association of miRNAs with genes with trinucleotide repeats for recommending for the diagnosis of nucleotide repeat expansion disorders.

scholarly journals microCT-Based Phenomics in the Zebrafish Skeleton Reveals Virtues of Deep Phenotyping in a Distributed Organ System

2017 ◽  
Author(s):  
Matthew Hur ◽  
Charlotte A. Gistelinck ◽  
Philippe Huber ◽  
Jane Lee ◽  
Marjorie H. Thompson ◽  
...  
Keyword(s):  
Axial Skeleton ◽  
Thyroid Stimulating Hormone ◽  
Effect Sizes ◽  
Genetic Screens ◽  
Spatially Distributed ◽  
Functional Understanding ◽  
Increase Productivity ◽  
Brittle Bone Disease ◽  
Mutant Phenotypes ◽  
Deep Phenotyping

ABSTRACTPhenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

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