One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical ...compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 μm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused ...by mutations in the highly conserved
gene. Here, we generated
morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant
larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for
disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for
Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic
disease.
Abstract
Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests ...that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of ATP13A2-associated disorders is not yet well understood and currently, these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for ATP13A2 deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. In this study, we show that the deletion of YPK9 (the yeast orthologue of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish—N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new in vivo model for investigating ATP13A2 deficiency.
PDF
