Abstract 229: Searching Genetic Modifiers For bag3 -based Cardiomyopathy Using Adult Zebrafish Models

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Yonghe Ding ◽  
Alexey Dvornikov ◽  
Xiao Ma ◽  
Hong Zhang ◽  
Xiaolei Xu
Keyword(s):  
Cardiac Dysfunction ◽  
Gene Knockout ◽  
Individualized Medicine ◽  
Genetic Modifier ◽  
Fish Mortality ◽  
Genetic Modifiers ◽  
Adult Zebrafish ◽  
Transcription Activator ◽  
Inherited Cardiomyopathy ◽  
Forward Mutagenesis

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3 (bag3) gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the bag3-/- fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed ex vivo assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on bag3 -based cardiomyopathy model. Different from the other three GBT mutants, GBT0411+/- , which tags the long isoform of dnajb6 b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the bag3-/- fish, suggesting dnajb6b as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the GBT0411+/-;bag3-/- double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.

Abstract 41: Dnajb6b Is A Novel Genetic Modifier For Cardiomyopathy That Regulates ER Stress Response

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Yonghe Ding ◽  
Weibin Liu ◽  
Beninio Gore ◽  
Stephen C Ekker ◽  
Xiaolei Xu
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Insertional Mutagenesis ◽  
Genetic Modifier ◽  
Phenotypic Expression ◽  
Modifier Genes ◽  
Genetic Modifiers ◽  
Loss Of Function ◽  
Adult Zebrafish ◽  
Er Stress Response

Background: Cardiomyopathy and heart failure affect millions of people worldwide. Because genetic modifiers contribute in large part to the highly variable phenotypic expression of cardiomyopathy in patients even with identical disease-causing mutations, the identification of modifier genes for this disease will greatly improve risk stratification, prognostic test development, and personalized therapy. However, only a rather limited number of modifier genes for cardiomyopathy have been identified sporadically. Objective: To identify genetic modifiers for cardiomyopathy using a novel insertional mutagenesis screening approach in adult zebrafish. Methods and Results: We screened 476 gene break-transposon (GBT) lines and isolated 44 zebrafish insertional cardiac (ZIC) mutants. Employing doxorubicin (DOX) stress to these ZIC mutants, we identified four candidate GBT lines that modified the progression of DOX-induced cardiomyopathy. Here, we report the detailed study of the GBT0411 mutant that exacerbated DOX-induced cardiomyopathy. GBT0411 mutant was tagged to the dnajb6b gene. Mutations in the short (sarcomeric) isoform of its human homologue gene DNAJB6 was recently reported to cause limb-girdle muscular dystrophy type 1D. Interestingly, our data showed that long (nuclei) isoform (dnajb6b[L]) was the major isoform expressed in the heart, and loss-of-function of which deteriorated the progression of DOX-induced cardiomyopathy. We further found that a cardiomyocyte-specific dnajb6b(L) transgene reverted the deleterious modifying effect of GBT0411 mutant, and exerted a cardioprotective function on chronic anemia induced cardiomyopathy. Mechanistically, Dnajb6b(L) could partially localize to endoplasmic reticulum (ER) upon ER stress, and function as an ER stress suppressor. Indeed, inhibition of ER stress by using a chemical chaperon mimics the cardioprotective effect of dnajb6b(L) transgene. Conclusions: By conducting an unbiased mutagenesis screening in adult zebrafish, we identified dnajb6b as a novel genetic modifier for cardiomyopathy. A cardioprotective function was identified by overexpressing its long isoform in cardiomyocytes, which might be conveyed by inhibition of ER stress response.

Abstract 381: Ex vivo Heart Function Analysis in Adult Zebrafish

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Alexey V Dvornikov ◽  
Hong Zhang ◽  
Xiaolei Xu
Keyword(s):  
Ex Vivo ◽  
Function Analysis ◽  
Heart Function ◽  
Heart Diseases ◽  
Genetic Modifiers ◽  
Adult Zebrafish ◽  
Inherited Cardiomyopathy ◽  
Novel Approach ◽  
Vertebrate Model

Zebrafish ( Danio rerio ) is an efficient vertebrate model of human cardiomyopathy which is amenable to the medium throughput screening approaches opening opportunities to search new genetic modifiers via mutagenesis screening and assessing compound-based therapies at larger scale. The advent of genome editing technology enables the generation of a panel of genetic models of cardiomyopathy with mutations in leading cardiomyopathy genes. However, one of the major bottlenecks for adult zebrafish as a cardiomyopathy model is the lack of appropriate cardiac functional assays. Due to small heart size, in vivo methods such as those based on echocardiography, are limited by their insufficient resolution. Here, we report the development of an ex vivo approach aimed to facilitate phenotyping in adult zebrafish. We show that our method is able to quantify parameters of pump function of the heart, including end-diastolic/systolic length/volumes, ejection and shortening fractions, and velocities of contraction/relaxation. We defined the basic parameters of these indices using different wild-type strains, age, and sex, and then demonstrated that our method can be useful in definition of progression of pathogenesis of both acquired (doxorubicin-injected) and inherited cardiomyopathy models. We conclude that our novel approach shall facilitate cardiac phenotyping in adult zebrafish models of heart diseases.

Genetic Modifiers of Tauopathy in Drosophila

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1233-1242
Author(s):  
Joshua M Shulman ◽  
Mel B Feany
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Molecular Mechanisms ◽  
Genetic Modifier ◽  
Genetic Modifiers ◽  
Protein Tau ◽  
Major Class ◽  
Drosophila Model ◽  
Tau Kinases

Abstract In Alzheimer's disease and related disorders, the microtubule-associated protein Tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles. Mutations in the tau gene cause familial frontotemporal dementia. To investigate the molecular mechanisms responsible for Tau-induced neurodegeneration, we conducted a genetic modifier screen in a Drosophila model of tauopathy. Kinases and phosphatases comprised the major class of modifiers recovered, and several candidate Tau kinases were similarly shown to enhance Tau toxicity in vivo. Despite some clinical and pathological similarities among neurodegenerative disorders, a direct comparison of modifiers between different Drosophila disease models revealed that the genetic pathways controlling Tau and polyglutamine toxicity are largely distinct. Our results demonstrate that kinases and phosphatases control Tau-induced neurodegeneration and have important implications for the development of therapies in Alzheimer's disease and related disorders.

scholarly journals Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atsuko Yamazaki ◽  
Shumpei Yamakawa ◽  
Yoshiaki Morino ◽  
Yasunori Sasakura ◽  
Hiroshi Wada
Keyword(s):  
Sea Urchins ◽  
Gene Knockout ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Regulatory System ◽  
Nutrient Sensing ◽  
Regulatory Genes ◽  
Transcription Activator ◽  
Patiria Pectinifera ◽  
Skeleton Formation

AbstractThe larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

ICAM-1 and VCAM-1 mediate endotoxemic myocardial dysfunction independent of neutrophil accumulation

2002 ◽  
Vol 283 (2) ◽  
pp. R477-R486 ◽  
Author(s):  
Christopher D. Raeburn ◽  
Casey M. Calkins ◽  
Michael A. Zimmerman ◽  
Yong Song ◽  
Lihua Ao ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Cardiac Dysfunction ◽  
Gene Knockout ◽  
Myocardial Dysfunction ◽  
Left Ventricular ◽  
Immunofluorescent Staining ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Neutrophil Accumulation

Both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been implicated in neutrophil-mediated lung and liver injury during sepsis. However, the role of these adhesion molecules as well as the contribution of neutrophils in myocardial dysfunction during sepsis remains to be determined. The purpose of this study was to examine the role of ICAM-1, VCAM-1, and neutrophils in lipopolysaccharide (LPS)-induced myocardial dysfunction. Mice were subjected to LPS (0.5 mg/kg ip) or vehicle (normal saline), and left ventricular developed pressure (LVDP) was determined by the Langendorff technique. LVDP was depressed by nearly 40% at 6 h after LPS. Immunofluorescent staining revealed a temporal increase in myocardial ICAM-1/VCAM-1 expression and neutrophils after LPS. Antibody blockade of VCAM-1 reduced myocardial neutrophil accumulation and abrogated LPS-induced cardiac dysfunction. Antibody blockade or absence of ICAM-1 (gene knockout) also abrogated LPS-induced cardiac dysfunction but did not reduce neutrophil accumulation. Neutrophil depletion (vinblastine or antibody) did not protect from LPS-induced myocardial dysfunction. Our results suggest that although endotoxemic myocardial dysfunction requires both ICAM-1 and VCAM-1, it occurs independent of neutrophil accumulation.

Genome Editing Toolbox

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-11-SCI-11
Author(s):  
Andrew M. Scharenberg
Keyword(s):  
Genome Engineering ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Practical Implementation ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Dna Breaks ◽  
Transcription Activator ◽  
Advisory Committees ◽  
Core Technology

Abstract Nucleases capable of making targeted breaks in genomic DNA are a core technology required for genome engineering, an emerging field of technology for making precise alterations in cellular genomes. Over the past ten years, four major platforms have emerged for generation of nucleases able to make targeted DNA breaks with a high degree of efficiency and specificity: homing endonucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and RNA-guided nucleases. This talk will cover the biochemistry and platform-specific attributes of each type of nuclease, along with evolution/improvements in nucleases and related technologies and aspects of the practical implementation of nuclease technology for gene knockout and gene repair in primary hematopoietic cells. Disclosures Scharenberg: Pregenen Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cellectis therapeutics: Consultancy.

337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES

2013 ◽  
Vol 25 (1) ◽  
pp. 316
Author(s):  
S. C. Fahrenkrug ◽  
W. Tan ◽  
S. G. Lillico ◽  
D. Stverakova ◽  
C. Proudfoot ◽  
...  
Keyword(s):  
Quantitative Trait ◽  
Gene Editing ◽  
Production Systems ◽  
Gene Knockout ◽  
Direct Injection ◽  
Genetic Enhancement ◽  
Transcription Activator ◽  
Tal Effectors ◽  
Cattle Breeds ◽  
Specific Allele

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

2015 ◽  
Vol 27 (1) ◽  
pp. 108
Author(s):  
H. Matsunari ◽  
M. Watanabe ◽  
K. Nakano ◽  
A. Uchikura ◽  
Y. Asano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Production Efficiency ◽  
Gene Knockout ◽  
Genetically Modified ◽  
Zinc Finger Nucleases ◽  
International Institute ◽  
Induced Mutations ◽  
Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants

2021 ◽  
pp. ASN.2020070991
Author(s):  
Ping Zhu ◽  
Qi Qiu ◽  
Peter C. Harris ◽  
Xiaolei Xu ◽  
Xueying Lin
Keyword(s):  
Renal Cysts ◽  
Cyst Formation ◽  
Adult Zebrafish ◽  
Transcription Activator ◽  
Primary Defect ◽  
Causative Gene ◽  
Mtor Inhibition ◽  
Adaptive Action ◽  
Single Nephron ◽  
Vertebrate Model

BackgroundAlthough zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.MethodsTranscription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67, the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity.ResultsAdult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants.ConclusionsAdult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.

scholarly journals PhenoModifier: a genetic modifier database for elucidating the genetic basis of human phenotypic variation

2019 ◽  
Author(s):  
Hong Sun ◽  
Yangfan Guo ◽  
Xiaoping Lan ◽  
Jia Jia ◽  
Xiaoshu Cai ◽  
...  
Keyword(s):  
Phenotypic Variation ◽  
Large Scale ◽  
Clinical Decision Making ◽  
Genetic Interaction ◽  
Genetic Modifier ◽  
Modifier Genes ◽  
Genetic Modifiers ◽  
Sequencing Data ◽  
Comprehensive Overview ◽  
Full Spectrum

Abstract From clinical observations to large-scale sequencing studies, the phenotypic impact of genetic modifiers is evident. To better understand the full spectrum of the genetic contribution to human disease, concerted efforts are needed to construct a useful modifier resource for interpreting the information from sequencing data. Here, we present the PhenoModifier (https://www.biosino.org/PhenoModifier), a manually curated database that provides a comprehensive overview of human genetic modifiers. By manually curating over ten thousand published articles, 3078 records of modifier information were entered into the current version of PhenoModifier, related to 288 different disorders, 2126 genetic modifier variants and 843 distinct modifier genes. To help users probe further into the mechanism of their interested modifier genes, we extended the yeast genetic interaction data and yeast quantitative trait loci to the human and we also integrated GWAS data into the PhenoModifier to assist users in evaluating all possible phenotypes associated with a modifier allele. As the first comprehensive resource of human genetic modifiers, PhenoModifier provides a more complete spectrum of genetic factors contributing to human phenotypic variation. The portal has a broad scientific and clinical scope, spanning activities relevant to variant interpretation for research purposes as well as clinical decision making.

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