Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach

Scavizzi, Ferdinando; Ryder, Edward; Newman, Stuart; Raspa, Marcello; Gleeson, Diane; Wardle‐Jones, Hannah; Montoliu, Lluı́s; Fernández, Almudena; Dessain, Marie-Laure; Larrigaldie, Vanessa; Khorshidi, Zuzana; Vuolteenaho, Reetta; Soininen, Raija; André, Philippe; Jacquot, Sylvie; Yi, Hong; Angelis, Martin Hrabě de; Ramirez-Solis, Ramiro; Doe, Brendan

doi:10.1007/s11248-015-9897-1

Cited by 22 publications

(12 citation statements)

References 16 publications

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“…On embryonic day 2.5 (E2.5), morulae were flushed from the uterus in accordance with a previously described protocol [18]. Embryos that were grown overnight in a microculture drop were transferred onto a gelatinised 24-well plate, each into its own well with 300 µl of embryonic stem cell medium, and left to grow for 10-12 days, after which DNA was extracted, as described [22].…”

Section: Nhlrc2 Knockout Mice (See Electronic Supplementary Material)mentioning

confidence: 99%

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease

et al. 2018

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A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

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Section: Nhlrc2 Knockout Mice (See Electronic Supplementary Material)mentioning

confidence: 99%

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease

et al. 2018

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“…DNA was extracted from blastocysts as published by (84). Briefly, single embryos were supplemented with 10 µl embryo lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl 2 , 0.1 mg/ml gelatin, 0.45% (v/v) NP40, 0.45% (v/v) Tween-20, 0.1 mg/ml proteinase K) and incubated at 56°C for 30 min, followed by 95°C for 10 min.…”

Section: Genotyping Of Blastocystsmentioning

confidence: 99%

Prm2deficiency triggers a Reactive Oxygen Species (ROS)-mediated destruction cascade during epididymal sperm maturation in mice

Schneider

Shakeri

Tröetschel

et al. 2020

Preprint

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Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Prm2-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm`s antioxidant capacity, indicated by downregulation of key proteins like SOD1 and PRDX5, ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stressmediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination. Significance statementSexual reproduction requires the successful fertilization of female eggs by male sperm. The generation of functional sperm is a complex, multi-step differentiation process known as spermatogenesis that takes places in the male testis. One important step for physiological sperm function is the incorporation of small proteins, known as protamines into the DNA.Defects within this process are common causes of male infertility. However, the underlying molecular mechanisms still remain largely unknown, thus preventing targeted therapies.Here, we identify the molecular cascade being initiated in protamine-deficient murine sperm that ultimately impedes fertilization. Our findings have broad implications for the development of new treatment options for infertile men with faulty protamination that seek medical advice.

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“…blastocysts. This will allow genotyping to be performed on these blastocysts and give a comparison of injected vs. non-injected developmental rates [27].…”

Section: Experimental Designmentioning

confidence: 99%

Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector

Doe

Brown

Boroviak

2018

MPs

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2-4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection.

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Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach

Cited by 22 publications

References 16 publications

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease

Prm2deficiency triggers a Reactive Oxygen Species (ROS)-mediated destruction cascade during epididymal sperm maturation in mice

Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector

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