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'ta Hücresel Onarım Stratejilerini Araştırmak için Retina Dejenerasyonu Oluşturma

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.

Yazar Spotlight: Xenopus Kurbağasında Retinal Yenilenme Mekanizmalarını Keşfetmek 

Xenopus Kurbağa Yavrularında Retina Yaralanma Modellerinin Oluşturulması

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 Aracılı Rodopsin Nakavtı ile Xenopus Kurbağa Yavrularında Çubuk Hücre Dejenerasyonu

Başlamak için, keskinleştirilmiş bir kılcal damar alın, 2 ila 10 mikrolitre CRISPR Cas9 ribonükleoprotein kompleksi veya kontrol solüsyonu yüklemek için bir mikro yükleyici ucu kullanın. Daha sonra, doldurulmuş kılcal damarı mikro enjektör işleyicisine yerleştirin. Ejeksiyon hacmini 10 nanolitrede ayarlamak için kılcal ucu ince forseps ile kırın.Bir hücre aşaması embriyosunu% 3 polisakroz çözeltisi ile 100 milimetrelik bir Petri kabına yapıştırılmış bir ızgaraya yerleştirin. Mikro enjektörü kullanarak ve bir stereo mikroskop altında, 10 nanolitre çözeltiyi kortikal bölgeye, özellikle sitoplazmik zarın altındaki hayvan kutbu seviyesine enjekte edin. 24 saat sonra, floresan stereo mikroskop altında floresein, lizin, dekstrin görselleştirin ve iyi enjekte edilmiş rho gevrek embriyoları seçin.Rho gevrek kurbağa yavrusu retinalarının kaspaz 3 etiketlemesi, bazı çubukların apoptoza uğradığını gösterdi. Histolojik boyama, nükleer tabakaların küresel olarak korunduğunu, ancak fotoreseptör dış segmentlerinin ciddi şekilde kısaldığını ortaya çıkardı. Fotoreseptör belirteçleri ile yapılan daha ileri immün boyama analizi, çubuk dış segmentlerinin dejenerasyonunu gösterdi.

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 ...

Gelişim Biyolojisi

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Retinal Rejenerasyon Mekanizmalarını İncelemek için Xenopus Kurbağa Yavrularında Mekanik Retina Ponksiyonu

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 Sistemi Kullanılarak Xenopus Kurbağa Yavrularında Koşullu Çubuk Hücre Ablasyonu

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Sitotoksik Göz İçi CoCl2 Enjeksiyonları ile Xenopus Kurbağa Yavrularında Retina Hücre Dejenerasyonu