scholarly journals Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish

Author(s):  
Rachael E. Quick ◽  
Luke D. Buck ◽  
Sweta Parab ◽  
Zane R. Tolbert ◽  
Ryota L. Matsuoka

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.

2020 ◽  
Vol 27 (34) ◽  
pp. 5790-5828 ◽  
Author(s):  
Ze Wang ◽  
Chunyang He ◽  
Jing-Shan Shi

Neurodegenerative diseases are a heterogeneous group of disorders characterized by the progressive degeneration of the structure and function of the central nervous system or peripheral nervous system. Alzheimer's Disease (AD), Parkinson's Disease (PD) and Spinal Cord Injury (SCI) are the common neurodegenerative diseases, which typically occur in people over the age of 60. With the rapid development of an aged society, over 60 million people worldwide are suffering from these uncurable diseases. Therefore, the search for new drugs and therapeutic methods has become an increasingly important research topic. Natural products especially those from the Traditional Chinese Medicines (TCMs), are the most important sources of drugs, and have received extensive interest among pharmacist. In this review, in order to facilitate further chemical modification of those useful natural products by pharmacists, we will bring together recent studies in single natural compound from TCMs with neuroprotective effect.


2002 ◽  
Vol 22 (12) ◽  
pp. 4147-4157 ◽  
Author(s):  
Eleni Goshu ◽  
Hui Jin ◽  
Rachel Fasnacht ◽  
Mike Sepenski ◽  
Jacques L. Michaud ◽  
...  

ABSTRACT The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity.


2015 ◽  
Vol 34 (4) ◽  
pp. 300-307 ◽  
Author(s):  
Swati Omanwar ◽  
M. Fahim

Vascular endothelium plays a vital role in the organization and function of the blood vessel and maintains homeostasis of the circulatory system and normal arterial function. Functional disruption of the endothelium is recognized as the beginning event that triggers the development of consequent cardiovascular disease (CVD) including atherosclerosis and coronary heart disease. There is a growing data associating mercury exposure with endothelial dysfunction and higher risk of CVD. This review explores and evaluates the impact of mercury exposure on CVD and endothelial function, highlighting the interplay of nitric oxide and oxidative stress.


eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Catherine M Drerup ◽  
Amy L Herbert ◽  
Kelly R Monk ◽  
Alex V Nechiporuk

Mitochondrial transport in axons is critical for neural circuit health and function. While several proteins have been found that modulate bidirectional mitochondrial motility, factors that regulate unidirectional mitochondrial transport have been harder to identify. In a genetic screen, we found a zebrafish strain in which mitochondria fail to attach to the dynein retrograde motor. This strain carries a loss-of-function mutation in actr10, a member of the dynein-associated complex dynactin. The abnormal axon morphology and mitochondrial retrograde transport defects observed in actr10 mutants are distinct from dynein and dynactin mutant axonal phenotypes. In addition, Actr10 lacking the dynactin binding domain maintains its ability to bind mitochondria, arguing for a role for Actr10 in dynactin-mitochondria interaction. Finally, genetic interaction studies implicated Drp1 as a partner in Actr10-dependent mitochondrial retrograde transport. Together, this work identifies Actr10 as a factor necessary for dynactin-mitochondria interaction, enhancing our understanding of how mitochondria properly localize in axons.


Development ◽  
1994 ◽  
Vol 1994 (Supplement) ◽  
pp. 61-77 ◽  
Author(s):  
J. Robert Manak ◽  
Matthew P. Scott

Dramatic successes in identifying vertebrate homeobox genes closely related to their insect relatives have led to the recognition of classes within the homeodomain superfamily. To what extent are the homeodomain protein classes dedicated to specific functions during development? Although information on vertebrate gene functions is limited, existing evidence from mice and nematodes clearly supports conservation of function for the Hox genes. Less compelling, but still remarkable, is the conservation of other homeobox gene classes and of regulators of homeotic gene expression and function. It is too soon to say whether the cases of conservation are unique and exceptional, or the beginning of a profoundly unified view of gene regulation in animal development. In any case, new questions are raised by the data: how can the differences between mammals and insects be compatible with conservation of homeobox gene function? Did the evolution of animal form involve a proliferation of new homeodomain proteins, new modes of regulation of existing gene types, or new relationships with target genes, or is evolutionary change largely the province of other classes of genes? In this review, we summarize what is known about conservation of homeobox gene function.


2019 ◽  
Vol 32 (11) ◽  
pp. 1207-1215
Author(s):  
Babak Emamalizadeh ◽  
Yousef Daneshmandpour ◽  
Abbas Tafakhori ◽  
Sakineh Ranji-Burachaloo ◽  
Sajad Shafiee ◽  
...  

Abstract Background X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the ABCD1 gene located on Xq28. X-ALD is characterized by a spectrum of different manifestations varying in patients and families. Methods Four pedigrees with X-ALD consisting of patients and healthy members were selected for investigation of ABCD1 gene mutations. The mutation analysis was performed by polymerase chain reaction (PCR) followed by direct sequencing of all exons. The identified mutations were investigated using bioinformatics tools to predict their effects on the protein product and also to compare the mutated sequence with close species. Results One previously known missense mutation (c.1978 C > T) and three novel mutations (c.1797dupT, c.879delC, c.1218 C > G) were identified in the ABCD1 gene, each in one family. Predicting the effects of the mutations on protein structure and function indicated the probable damaging effect for them with significant alterations in the protein structure. We found three novel mutations in the ABCD1 gene with damaging effects on its protein product and responsible for X-ALD.


2001 ◽  
Vol 14 (3) ◽  
pp. 426-430 ◽  
Author(s):  
Bruno Dombrecht ◽  
Jos Vanderleyden ◽  
Jan Michiels

The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported. Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp. NGR234. The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences. Vector derivatives with the constitutive nptII promoter or a promoter-less gusA gene are suitable for the study of gene function or regulation in bacteria.


2009 ◽  
Vol 89 (1) ◽  
pp. 1-26 ◽  
Author(s):  
Benjamin Turgeon ◽  
Sylvain Meloche

The mouse represents the model of choice to study the biological function of mammalian genes through mutation of its genome. However, the biggest challenge of mouse geneticists remains the phenotypic analysis of mouse mutants. A survey of mouse mutant databases reveals a surprisingly high number of gene mutations leading to neonatal death. These genetically modified mouse mutants have been instrumental in elucidating gene function and have become important models of congenital human diseases. The main complication when phenotyping mutant mice dying during the neonatal period is the large spectrum of physiological systems whose defects can challenge neonatal survival. Here, we present a comprehensive review of gene mutations leading to neonatal lethality and discuss the impact of these mutations on the major physiological processes critical to mouse newborn survival: parturition, breathing, suckling, and homeostasis. Selected examples of mouse mutants are highlighted to illustrate how the precise identification of the timing and cause of death associated with these physiological processes allows for a more profound understanding of the underlying cellular and molecular defects. This review provides a guide for the analysis of neonatal lethal phenotypes in mutant mice that will be helpful for dissecting out the function of specific genes during mouse development.


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