Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A&gt;G Mutation in<i>USH2A</i>

Slijkerman, Ralph; Goloborodko, Alexander; Broekman, Sanne; Vrieze, Erik de; Hetterschijt, Lisette; Peters, Theo; Gerits, Milou; Kremer, Hannie; Wijk, Erwin van

doi:10.1089/zeb.2018.1613

Cited by 22 publications

(22 citation statements)

References 33 publications

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“… (a) Injection of MO- upf 1 (splice-blocking morpholino) results in the partial skipping of upf 1 exon 1, leading to a frame shift, a premature stop codon and the premature termination of Upf1 translation [ 43 , 44 ]. (b) Injection of MO- bag2 (splice-blocking morpholino) results in the integration of the intron 2, a frame shift, a premature stop codon and the premature termination of Bag2 translation.…”

Section: Supporting Informationmentioning

confidence: 99%

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency

et al. 2020

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Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart failure, as well as myofibrillar myopathy (MFM), which is characterized by protein aggregation and myofibrillar disintegration in skeletal muscle cells. Here, we generated a CRISPR/Cas9-induced Bag3 knockout zebrafish line and found the complete preservation of heart and skeletal muscle structure and function during embryonic development, in contrast to morpholino-mediated knockdown of Bag3. Intriguingly, genetic compensation, a process of transcriptional adaptation which acts independent of protein feedback loops, was found to prevent heart and skeletal muscle damage in our Bag3 knockout model. Proteomic profiling and quantitative real-time PCR analyses identified Bag2, another member of the Bag protein family, significantly upregulated on a transcript and protein level in bag3 -/- mutants. This implied that the decay of bag3 mutant mRNA in homozygous bag3 -/- embryos caused the transcriptional upregulation of bag2 expression. We further demonstrated that morpholino-mediated knockdown of Bag2 in bag3 -/- embryos evoked severe functional and structural heart and skeletal muscle defects, which are similar to Bag3 morphants. However, Bag2 knockdown in bag3 +/+ or bag3 +/- embryos did not result in (cardio-)myopathy. Finally, we found that inhibition of the nonsense-mediated mRNA decay (NMD) machinery by knockdown of upf1 , an essential NMD factor, caused severe heart and skeletal muscle defects in bag3 -/- mutants due to the blockade of transcriptional adaptation of bag2 expression. Our findings provide evidence that genetic compensation might vitally influence the penetrance of disease-causing bag3 mutations in vivo .

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Section: Supporting Informationmentioning

confidence: 99%

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency

et al. 2020

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“…To increase the efficiency of the homology directed repair mechanism, the nonhomologous end joining repair mechanism was inhibited by a splice-blocking morpholino targeting ku70 (5 -AACTTTTTAGGCTCACCTGCATAGT-3 ). 19,20 A mix of 1 nL containing donor template (0.3 μM), gRNA (600 pg), Cas9 protein (5 ng), ku70 morpholino (1.5 ng), phenol-red (0.01%), and KCl 0.2 M was injected using a Pneumatic Picopump pv280 (World Precision Instruments) into zebrafish embryos at a one-cell stage. The injected embryos were grown into adulthood at 28°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 , supplemented with 0.01% methylene blue).…”

Section: Generation Of Znf408 Zebrafish Modelmentioning

confidence: 99%

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature

Karjosukarso

Ali

Peters

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR. METHODS. Using CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked. RESULTS. Intriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf. CONCLUSIONS. Together, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina.

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“…the molecular targets of the root gene should be the same in the model and in the human, as should the regulation and downstream molecular effects of the disease-causing gene variant. This is always a concern in model systems that may be less complex than the human, even when a model is ‘humanized’ to carry the human gene sequence, sometimes with the precise disease-associated mutation (Slijkerman et al, 2018). For a rare disorder like cystic fibrosis, the conservation of the entire functional pathway and affected organ systems between human and mice is very high, making treatments in the mouse an accurate representation of the likely response in the human (Knowles and Durie, 2002).…”

Section: Target Validitymentioning

confidence: 99%

From gene to treatment: supporting rare disease translational research through model systems

Hmeljak

Justice

2019

Disease Models &Amp; Mechanisms

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Individual rare diseases may affect only a few people, making them difficult to recognize, diagnose or treat by studying humans alone. Instead, model organisms help to validate genetic associations, understand functional pathways and develop therapeutic interventions for rare diseases. In this Editorial, we point to the key parameters in face, construct, predictive and target validity for accurate disease modelling, with special emphasis on rare disease models. Raising the experimental standards for disease models will enhance successful clinical translation and benefit rare disease research.

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Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G Mutation inUSH2A

Cited by 22 publications

References 33 publications

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature

From gene to treatment: supporting rare disease translational research through model systems

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