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

Xenopusの細胞修復戦略を調査するための網膜変性の生成

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.

著者スポットライト:Xenopus Frogの網膜再生メカニズムの探索 

Xenopusオタマジャクシにおける網膜損傷モデルの生成

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  

CRISPR/Cas9を介したロドプシンノックアウトによるツコノプスオタマジャクシの桿体細胞変性

まず、鋭利なキャピラリーを取り、マイクロローダーチップを使用して、2〜10マイクロリットルのCRISPR Cas9リボ核タンパク質複合体またはコントロール溶液をロードします。その後、充填したキャピラリーをマイクロインジェクターハンドラーに入れます。細い鉗子で毛細血管先端を折って吐出量を10ナノリットルに調整します。1細胞期の胚を、3%ポリスクロース溶液を含む100ミリメートルのペトリ皿に接着したグリッド上に配置します。マイクロインジェクターを使用し、実体顕微鏡下で、10ナノリットルの溶液を皮質領域、特に細胞質膜の下の動物極レベルに注入します。24時間後、フルオレセイン、リジン、デキストリンを蛍光実体顕微鏡で可視化し、適切に注入されたrho crispant胚を選択します。rho crispant オタマジャクシ網膜のカスパーゼ 3 標識は、一部の桿体がアポトーシスを起こすことを示しました。組織学的染色により、核層は全体的に保存されているが、視細胞の外側のセグメントは著しく短縮していることが明らかになった。視細胞マーカーを用いたさらなる免疫染色分析により、桿体外側セグメントの変性が示されました。

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 ...

発生生物学

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Xenopusオタマジャクシの網膜再生機構を研究するための機械的網膜穿刺

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

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

NTR-MTZシステムを用いたツ コツメガ ネオタマジャクシの条件付き桿体細胞アブレーション

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

細胞傷害性眼内CoCl2注射によるXenopusオタマジャクシの網膜細胞変性