Name: Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout at JoVE.com

rod cell degeneration in xenopus tadpoles by crispr/cas9-mediated rhodopsin knockout

Génération de la dégénérescence rétinienne pour étudier les stratégies de réparation cellulaire chez le xénope

Millions of people worldwide are afflicted with various retinal degenerative diseases leading to blindness, such as retinitis pigmentosa, diabetic retinopathy, or age-related macular degeneration (AMD). To date, these conditions remain largely untreatable. Current therapeutic approaches under evaluation include gene therapy, cell or tissue transplantations, neuroprotective treatments, optogenetics, and prosthetic devices. Another emerging strategy is based on self-regeneration through the activation of endogenous cells with stem cell potential. M&#252;ller glial cells, the major glial cell type of the retina, are among cellular sources of interest in this context. Upon injury, they can dedifferentiate, proliferate, and generate neurons1,2,3. Although this process is very effective in zebrafish or Xenopus, it is largely inefficient in mammals.
Nonetheless, it has been shown that appropriate treatments with mitogenic proteins or overexpression of various factors can induce mammalian M&#252;ller glia cell-cycle re-entry and, in some cases, trigger their subsequent&#160;neurogenesis commitment1,2,3,4,5. This remains, however, largely insufficient for treatments. Hence, increasing our knowledge of the molecular mechanisms underlying regeneration is necessary to identify molecules able to efficiently turn M&#252;ller stem-like cell properties into new cellular therapeutic strategies.
With this aim, we developed several injury paradigms in Xenopus that trigger retinal cell degeneration. Here, we present (1) a mechanical retinal injury that is not cell type-specific, (2) a conditional and reversible cell ablation model using the NTR-MTZ system that targets rod cells, (3) a CRISPR/Cas9-mediated rhodopsin knockout, a model of retinitis pigmentosa that triggers progressive rod cell degeneration, and (4) a CoCl2-induced cytotoxic model that according to the dose can specifically target cones or lead to broader retinal cell degeneration. We highlight the particularities, advantages, and disadvantages of each paradigm.

Pleins feux sur l’auteur : Explorer les mécanismes de régénération rétinienne chez la grenouille xénope 

Génération de modèles de lésions rétiniennes chez les têtards Xénope

My lab works on retinal regeneration. We are using the Xenopus frog as a model system. This amphibian is indeed quite fascinating because unlike mammals, like us, it can regenerate its retina very efficiently in case of injury.And we are studying the underlying mechanism because, in the future, it could be useful to trigger retinal regeneration in human patients afflicted with retinal neurodegenerative diseases. We have recently discovered that in addition to stem cells in the periphery of the retina and the retinal pigmented epithelium, neuroglial cells can also be recruited for retinal regeneration in case of injury. And so we are now studying the links between neuroinflammation and the regenerative capacity of these cells.Indeed, it seems that the neuroinflammatory niche is a key player in the modulation of the regeneration of the retina. To study the cellular and molecular mechanisms involved in retinal regeneration, we developed several retinal injury paradigms in Xenopus. The first is a mechanical retinal injury.The second is a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation. The third is a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and, to finish, a cytotoxic model driven by intraocular injection of cobalt chloride or CoCl2. My host laboratory has showed that although Xenopus can regenerate its retina, the efficiency is highly variable and depends on the stages of the tadpole or on species, Xenopus laevis or tropicalis.This makes Xenopus a fantastic model for illustrating the molecular mechanisms that trigger or limit retinal regeneration.

Watch this Scientific Journal Video about Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout at JoVE.com

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout  

Dégénérescence des cellules en bâtonnets chez les têtards de Xénope par knock-out de la rhodopsine médiée par CRISPR/Cas9

Pour commencer, prenez un capillaire aiguisé, utilisez une pointe de micro-chargeur pour charger 2 à 10 microlitres du complexe ribonucléoprotéique CRISPR Cas9 ou de la solution témoin. Ensuite, placez le capillaire rempli dans le manipulateur de micro-injecteurs. Cassez l’embout capillaire à l’aide d’une pince fine pour ajuster le volume d’éjection à 10 nanolitres.Positionner les embryons d’un stade cellulaire sur une grille collée dans une boîte de Pétri de 100 millimètres avec une solution de polysaccharose à 3 %. À l’aide du micro-injecteur, et sous un stéréomicroscope, injecter 10 nanolitres de la solution dans la région corticale, plus précisément au niveau du pôle animal sous la membrane cytoplasmique. Après 24 heures, visualisez la fluorescéine, la lysine, la dextrine sous un stéréomicroscope fluorescent et sélectionnez des embryons rho crispant bien injectés.Le marquage à la caspase 3 des rétines têtardes rho crispant a montré que certains bâtonnets subissent une apoptose. La coloration histologique a révélé une préservation globale des couches nucléaires, mais un raccourcissement sévère des segments externes des photorécepteurs. D’autres analyses de marquage immunologique avec des marqueurs photorécepteurs ont montré la dégénérescence des segments externes des bâtonnets.

Research

JoVE Journal

Developmental Biology

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

Recherche

Biologie du développement

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Ponction mécanique de la rétine chez les têtards de Xénope pour étudier les mécanismes de régénération de la rétine

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System  

Ablation conditionnelle des cellules en bâtonnets chez les têtards de Xénope à l’aide du système NTR-MTZ

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Dégénérescence des cellules rétiniennes chez les têtards de Xénope par injections intraoculaires cytotoxiques de CoCl2