CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

ABSTRACTGenomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.

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CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

PLoS ONE ◽

10.1371/journal.pone.0261331 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261331

Author(s):

Laurie J. Mitchell ◽

Valerio Tettamanti ◽

Justin S. Rhodes ◽

N. Justin Marshall ◽

Karen L. Cheney ◽

...

Keyword(s):

Gene Editing ◽

Mutant Gene ◽

Reef Fishes ◽

Loss Of Function ◽

Social Hierarchies ◽

Sequential Hermaphroditism ◽

Starting Point ◽

Pattern Development ◽

Encoding Gene ◽

Biallelic Mutations

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

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Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis elegans embryo.

Genetics ◽

10.1093/genetics/126.3.593 ◽

1990 ◽

Vol 126 (3) ◽

pp. 593-605 ◽

Cited By ~ 5

Author(s):

P E Mains ◽

K J Kemphues ◽

S A Sprunger ◽

I A Sulston ◽

W B Wood

Keyword(s):

Caenorhabditis Elegans ◽

Maternal Effect ◽

Mutant Gene ◽

Temperature Sensitive ◽

Loss Of Function ◽

Gain Of Function ◽

Lethal Mutations ◽

Cell Embryo ◽

New Gene ◽

The One

Abstract We describe interactions between maternal-effect lethal mutations in four genes of Caenorhabditis elegans whose products appear to be involved in the meiotic and mitotic divisions of the one-cell embryo. Mitosis is disrupted by two dominant temperature-sensitive gain-of-function maternal-effect lethal mutations, mei-1(ct46) and mel-26(ct61), and by recessive loss-of-function maternal-effect lethal mutations of zyg-9. The phenotypic defects resulting from these mutations are similar. Doubly mutant combinations show a strong enhancement of the maternal-effect lethality under semipermissive conditions, suggesting that the mutant gene products interact. We isolated 15 dominant suppressors of the gain-of-function mutation mei-1(ct46). Thirteen of these suppressors are apparently intragenic, but 11 of them suppress in trans as well as cis. Two extragenic suppressors define a new gene, mei-2. The suppressor mutations in these two genes also result in recessive maternal-effect lethality, but with meiotic rather than mitotic defects. Surprisingly, most of these suppressors are also able to suppress mel-26(ct61) in addition to mei-1(ct46). The products of the four genes mei-1, mei-2, zyg-9 and mel-26 could be responsible for some of the specialized features that distinguish the meiotic from the mitotic divisions in the one-cell embryo.

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A Relativistic Newtonian Mechanics Predicts with Precision the Results of Recent Neutrino-Velocity Experiments

JOURNAL OF ADVANCES IN PHYSICS ◽

10.24297/jap.v6i1.1824 ◽

2014 ◽

Vol 6 (1) ◽

pp. 1032-1035 ◽

Cited By ~ 1

Author(s):

Ramzi Suleiman

Keyword(s):

Null Hypothesis ◽

Experimental Studies ◽

Theoretical Models ◽

Newtonian Mechanics ◽

Speed Of Light ◽

Symmetry Principle ◽

Proposed Model ◽

Significant Difference ◽

The One ◽

Complete Collapse

The research on quasi-luminal neutrinos has sparked several experimental studies for testing the "speed of light limit" hypothesis. Until today, the overall evidence favors the "null" hypothesis, stating that there is no significant difference between the observed velocities of light and neutrinos. Despite numerous theoretical models proposed to explain the neutrinos behavior, no attempt has been undertaken to predict the experimentally produced results. This paper presents a simple novel extension of Newton's mechanics to the domain of relativistic velocities. For a typical neutrino-velocity experiment, the proposed model is utilized to derive a general expression for . Comparison of the model's prediction with results of six neutrino-velocity experiments, conducted by five collaborations, reveals that the model predicts all the reported results with striking accuracy. Because in the proposed model, the direction of the neutrino flight matters, the model's impressive success in accounting for all the tested data, indicates a complete collapse of the Lorentz symmetry principle in situation involving quasi-luminal particles, moving in two opposite directions. This conclusion is support by previous findings, showing that an identical Sagnac effect to the one documented for radial motion, occurs also in linear motion.

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COMPUTATIONAL AND EXPERIMENTAL STUDIES OF A LOW-EMISSION BURNER FOR A PERSPECTIVE TURBOJET ENGINE WITH THE COMPRESSION RATIO EXCEEDING 40

NONEQUILIBRIUM PROCESSES: RECENT ACCOMPLISHMENTS ◽

10.30826/nepcap9a-39 ◽

2020 ◽

Author(s):

M. A. Danilov ◽

◽

M. V. Drobysh ◽

A. N. Dubovitsky ◽

F. G. Markov ◽

...

Keyword(s):

Compression Ratio ◽

Experimental Studies ◽

The Other ◽

Aircraft Engines ◽

Engine Efficiency ◽

Other Hand ◽

Turbojet Engine ◽

Low Emission ◽

Civil Aircraft ◽

The One

Restrictions of emissions for civil aircraft engines, on the one hand, and the need in increasing the engine efficiency, on the other hand, cause difficulties during development of low-emission combustors for such engines.

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par-4, a gene required for cytoplasmic localization and determination of specific cell types in Caenorhabditis elegans embryogenesis.

Genetics ◽

10.1093/genetics/130.4.771 ◽

1992 ◽

Vol 130 (4) ◽

pp. 771-790 ◽

Cited By ~ 5

Author(s):

D G Morton ◽

J M Roos ◽

K J Kemphues

Keyword(s):

Caenorhabditis Elegans ◽

Cell Types ◽

Intestinal Cells ◽

Specific Cell ◽

Cytoplasmic Localization ◽

Loss Of Function ◽

Embryo Viability ◽

Cell Stage ◽

Maternal Genome

Abstract Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.

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Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Reproduction ◽

10.1530/rep.1.00297 ◽

2004 ◽

Vol 128 (3) ◽

pp. 301-311 ◽

Cited By ~ 193

Author(s):

Paolo Rinaudo ◽

Richard M Schultz

Keyword(s):

Gene Expression ◽

Preimplantation Embryos ◽

Expression Analysis Systematic Explorer ◽

Cell Stage ◽

Global Pattern ◽

Preimplantation Mouse Embryos ◽

Preimplantation Mouse ◽

Global Patterns ◽

The One

Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.

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Experimental studies on a mutant gene (cl) in the Mexican axolotl which affects cell membrane formation in embryos from females

Developmental Biology ◽

10.1016/0012-1606(73)90227-3 ◽

1973 ◽

Vol 32 (1) ◽

pp. 155-166 ◽

Cited By ~ 8

Author(s):

Carole R. Carroll ◽

E.B. Van Deusen

Keyword(s):

Cell Membrane ◽

Experimental Studies ◽

Mutant Gene ◽

Mexican Axolotl ◽

Membrane Formation

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Crashworthiness Performance of Mass-Efficient Extruded Structures

Transportation: Making Tracks for Tomorrow’s Transportation ◽

10.1115/imece2002-39077 ◽

2002 ◽

Cited By ~ 1

Author(s):

Robert R. Mayer ◽

Weigang Chen ◽

Anil Sachdev

Keyword(s):

Numerical Simulations ◽

Single Cell ◽

Experimental Testing ◽

Experimental Studies ◽

Nonlinear Material ◽

Dynamic Strain ◽

Cell Design ◽

Explicit Finite Element ◽

The One ◽

Crushing Behavior

Theoretical, numerical and experimental studies were conducted on the axial crushing behavior of traditional single-cell and innovative four-cell extrusions. Two commercial aluminum alloys, 6061 and 6063, both with two tempers (T4 and T6), were considered in the study. Testing coupons taken from the extrusions assessed the nonlinear material properties. A theoretical solution was available for the one-cell design, and was developed for the mean crushing force of the four-cell section. Numerical simulations were carried out using the explicit finite element code LS-DYNA. The aluminum alloy 6063T4 was found to absorb less energy than 6061T4, for both the one-cell and four-cell configurations. Both 6061 and 6063 in the T6 temper were found to have significant fracture in the experimental testing. Theoretical analysis and numerical simulations predicted a greater number of folds for the four-cell design, as compared to the one-cell design, and this was confirmed in the experiments. The theoretical improvement in energy absorption of 57% for the four-cell in comparison with the one-cell design was confirmed by experiment. The good agreement between the theoretical, numerical and experimental results allows confidence in the application of the theoretical and numerical tools for both single-cell and innovative four-cell extrusions. It was also demonstrated that these materials have very little dynamic strain rate effect.

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Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y02-093 ◽

2002 ◽

Vol 80 (7) ◽

pp. 618-624 ◽

Cited By ~ 1

Author(s):

P Jacquet ◽

J Buset ◽

J Vankerkom ◽

S Baatout ◽

L de Saint-Georges ◽

...

Keyword(s):

Calyculin A ◽

Embryonic Cells ◽

G2 Arrest ◽

Cell Stage ◽

Chromosome Fragments ◽

X Irradiation ◽

Radiation Induced ◽

Mouse Zygotes ◽

The One

PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3sigma pathway.Key words: PCC, embryo, oocyte, calyculin A, G2 arrest, cytokinesis.

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