Name: Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

conditional rod cell ablation in xenopus tadpoles using the ntr-mtz system

Xenopus의 세포 복구 전략을 조사하기 위한 망막 변성 생성

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.

저자 스포트라이트: Xenopus Frog의 망막 재생 메커니즘 탐구 

Xenopus 올챙이에서 망막 손상 모델 생성

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 Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

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

NTR-MTZ 시스템을 사용한 Xenopus 올챙이의 조건부 간상 세포 절제

먼저, 간상 광수용체 특이적 프로모터(rod photoreceptor specific promoter)에서 GFP와 니트로 환원효소(nitro reductase, NTR)를 발현하는 형질전환 계통을 사용합니다. NTR은 전구약물 메트로니다졸(MTZ)을 NTR 발현 간상체의 선택적 절제를 위한 세포독성제로 전환합니다. GFP 모니터링을 위해 숟가락을 사용하여 마취된 올챙이 한 마리를 부드럽게 잡고 작은 페트리 접시 안의 축축한 조직 위에 조심스럽게 놓습니다.형광 실체 현미경으로 GFP 형광을 관찰하고 눈의 사진 이미지를 캡처합니다. 올챙이와 함께 페트리 접시를 완전히 깨어날 때까지 기르는 물이 있는 큰 탱크에 조심스럽게 담그십시오. 망막 변성의 개별 모니터링을 위해 각 올챙이를 30ml의 10밀리몰 MTZ 또는 대조 용액의 형질전환 형제가 들어 있는 작은 용기에 넣습니다.일괄 처리를 위해 형질전환 올챙이를 1리터의 MTZ 또는 대조 용액과 함께 더 큰 용기에 넣습니다. 올챙이를 MTZ의 저하를 피하기 위해 어둠 속에서 섭씨 20도에서 일주일 동안 올챙이를 들어 올립니다. 7 일 후에, 형광성 입체 현미경의 밑에 GFP를 관찰하고 눈의 사진 이미지를 붙잡으십시오.형광 강도의 감소 또는 형광의 사실상 부재는 간상체의 열화를 나타냅니다. MTZ로 처리한 형질전환 올챙이의 눈은 대조군에 비해 GFP 형광의 감소를 보였으며, 이는 GFP 면역 염색에 의해 망막 절편에서 추가로 확인되었습니다.

Research

JoVE Journal

Developmental Biology

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

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 ...

연구

발생학

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

망막 재생 메커니즘을 연구하기 위한 Xenopus 올챙이의 기계적 망막 천자

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

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

CRISPR/Cas9-mediated Rhodopsin knockout에 의한 Xenopus 올챙이의 막대 세포 변성

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

세포독성 안구 내 CoCl2 주사에 의한 Xenopus 올챙이의 망막 세포 변성