eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Morpholino oligo design
NOTE: Sequences for A. mexicanus are available through National Center of Biotechnology Information (NCBI) Gene and NCBI SRA (https://www.ncbi.nlm.nih.gov), as well as from the Ensembl genome browser (https://www.ensembl.org). When designing a morpholino fo...

morpholino mediated gene editing: a gene knockdown technique using translational blocking morpholino oligonucleotides in single-celled cavefish eggs

Source: Stahl, B. A. et al., <a href="https://www.jove.com/v/59093">Manipulation of Gene Function in Mexican Cavefish.</a> J. Vis. Exp. (2019).
This video describes a gene knockdown technique using translational blocking morpholinos microinjected in the one-cell stage of the cavefish. Morpholinos are DNA oligonucleotides that bind to the complementary sequence of target mRNA and block its translation to a protein. Consequently, morpholinos are used as molecular tools to stop or reduce a gene&#8217;s expression transiently.

Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs

Source: Stahl, B. A. et al., Manipulation of Gene Function in Mexican Cavefish. J. Vis. Exp. (2019).
This video describes a gene knockdown technique using ...

Morpholinos are short, single-stranded redesigned oligonucleotides with differences in their phosphate backbone and sugar structure as compared to natural nucleotides. These features enhance morpholinos&#8217; stability at the physiological pH. Despite these variations, the morpholinos contain the standard nucleobases, which help in designing oligonucleotide complementary to the specific mRNA sequence from a desired target gene to be knocked down.
To perform morpholino-driven gene editing, begin by taking single-celled, cavefish eggs placed inside the wells of an agarose injection plate. Visualize the translucent and round eggs to locate the yolk sac - a fluid-filled structure present inside the egg.
Take a microinjection needle pre-filled with a solution containing designed morpholinos. Inject the solution directly into the egg yolk, ensuring morpholinos enter the cell cytoplasm. Inside the cells, the target gene forms the mRNA, which is transported to the cytoplasm.
Within the cytoplasm, the injected morpholino hybridizes with its complementary target mRNA transcript at the endogenous initiation site and extends beyond it. This process blocks the binding of the larger subunit of the ribosome with the nascent translation initiation complex consisting of small ribosomal subunit complexed with some specific proteins.
Subsequently, the progression of the translation initiation complex is sterically hindered, which stops the translation of mRNA into the protein. Finally, the target protein concentration reduces due to translational blocking by morpholinos.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Genome Editing Techniques

Gene regulation

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

JoVE Journal

Genetics

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs

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