KR102634464B1 - Method for producing animal model with retinal degeneration - Google Patents
Method for producing animal model with retinal degeneration Download PDFInfo
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- KR102634464B1 KR102634464B1 KR1020210067261A KR20210067261A KR102634464B1 KR 102634464 B1 KR102634464 B1 KR 102634464B1 KR 1020210067261 A KR1020210067261 A KR 1020210067261A KR 20210067261 A KR20210067261 A KR 20210067261A KR 102634464 B1 KR102634464 B1 KR 102634464B1
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- retinal degeneration
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Abstract
본 발명은 망막변성증 동물 모델의 제조 방법 및 이에 의해 제조된 망막변성증 동물 모델에 관한 것으로, 본 발명의 망막변성증 동물 모델은 인간의 눈과 유사하게 안구내 색소를 가지면서 망막 변성 양상을 나타내므로 망막변성증의 기전 연구에 유용하게 사용될 수 있다.The present invention relates to a method for producing a retinal degeneration animal model and a retinal degeneration animal model produced thereby. The retinal degeneration animal model of the present invention has intraocular pigment similar to that of a human eye and exhibits retinal degeneration patterns, so that the retina It can be useful in research on the mechanisms of degeneration.
Description
본 발명은 망막변성증 동물 모델의 제조 방법 및 이 방법에 의해 제조된 망막변성증 동물 모델에 관한 것이다.The present invention relates to a method for producing a retinal degeneration animal model and a retinal degeneration animal model produced by the method.
망막색소변성증은 망막색소와 관련된 유전자의 돌연변이로 인해 망막 내에 있는 광수용체 세포인 간상세포 또는 원추세포가 점진적으로 손상되어 시력이 손실되는 질환이다. 망막색소변성증은 다양한 원인 유전자의 이상에 의한 실명을 유발하는 망막 질환으로 10대 전후의 어린 나이부터 망막 변성이 서서히 진행되어 야맹증, 시야 장애 증상을 보이다가 심각한 시력 저하 진행으로 실명으로 이어지는 질환이다. 현재까지 망막색소변성증에 대한 표준 치료법은 개발된 바 없다. Retinitis pigmentosa is a disease that causes vision loss due to gradual damage to rods or cones, which are photoreceptor cells in the retina, due to mutations in genes related to retinal pigment. Retinitis pigmentosa is a retinal disease that causes blindness due to abnormalities in various causative genes. Retinal degeneration progresses slowly from a young age around the teenage years, leading to symptoms of night blindness and visual field problems, and then leading to blindness due to severe deterioration of vision. To date, no standard treatment for retinitis pigmentosa has been developed.
현재까지 표준 치료법이 없는 유전성 망막색소변성증에 대한 유전 이상 교정 유전자 치료제 혹은 세포 치료제를 이용한 치료법 개발을 위해서는 다양한 원인 유전자별 적절한 동물 모델의 확보가 필수적이다. 실험용 동물 모델 생산 및 판매 업체는 국내 및 전 세계적으로 많이 존재하지만 안과 질환에 특화된 실험 동물 생산 업체는 드물며, 안과적 이상을 보이는 동물을 보유하고 있더라도 실험 동물의 안과적 특성을 정확히 파악하고 평가하는 능력을 보유한 업체 역시 전무하여 특화된 능력을 보유한 연구자가 직접 개발하고 특성을 평가해야 한다.In order to develop treatment using gene therapy or cell therapy to correct genetic abnormalities for hereditary retinitis pigmentosa, for which there is no standard treatment to date, it is essential to secure appropriate animal models for various causative genes. Although there are many companies producing and selling laboratory animal models domestically and globally, there are few laboratory animal producers specializing in ophthalmic diseases, and even if they have animals showing ophthalmic abnormalities, they have the ability to accurately identify and evaluate the ophthalmic characteristics of laboratory animals. Since there are no companies with specialized capabilities, researchers with specialized capabilities must develop and evaluate the characteristics.
망막색소변성증의 동물 모델로는 마우스, 랫, 토끼, 돼지, 제브라피쉬 및 비인간영장류 등 다양한 사례가 있다. 그 중에서 마우스 모델이 비용이 저렴하고, 비교적 빠른 시간 내에 질병의 진행을 확인할 수 있으며, 유전자 조작이 가능하므로, 유전자 접근법을 통하여 인간 망막 질환의 기초가 되는 100개 이상의 유전자에 대한 마우스 모델이 연구된 바 있다.There are a variety of animal models for retinitis pigmentosa, including mice, rats, rabbits, pigs, zebrafish, and non-human primates. Among them, mouse models are inexpensive, can confirm disease progression in a relatively short period of time, and allow genetic manipulation, so mouse models for more than 100 genes that are the basis of human retinal diseases have been studied through genetic approaches. There is a bar.
인간망막변성증(Retinitis pigmentosa) 연구를 위하여 지난 10년 동안 가장 일반적, 보편적으로 사용된 마우스 모델은 PDE6B rd10 마우스 모델이며, 전 세계의 많은 연구소에서 연구에 활용되고 있다. PDE6B rd10 마우스 모델에서 망막생리학 및 병리생리학의 연구는 분자, 세포 및 조직 특이적 수준에서의 망막 발달, 유지 및 기능에 대한 이해를 도왔다. 해당 연구 결과는 인간의 망막변성증 원인 메커니즘을 확인하고, 광수용체 생존을 개선하고 유전자 대체에 의한 다양한 치료 접근법을 확립하는데 사용되어, 망막색소변성증으로 고통 받는 환자를 치료할 수 있는 효과적인 치료법 개발의 토대를 마련하였다.The most commonly used mouse model over the past 10 years to study human retinal degeneration (Retinitis pigmentosa) is the PDE6B rd10 mouse model, and it is being used in research in many research institutes around the world. Studies of retinal physiology and pathophysiology in the PDE6B rd10 mouse model have aided our understanding of retinal development, maintenance, and function at molecular, cellular, and tissue-specific levels. The research results were used to identify the causative mechanism of retinal degeneration in humans, improve photoreceptor survival, and establish various treatment approaches by gene replacement, laying the foundation for the development of effective treatments to treat patients suffering from retinitis pigmentosa. prepared.
이와 같이 망막변성 동물 모델 쥐는 랫보다 상대적으로 크기가 작은 마우스를 이용하여 개발된 모델이 대부분이며, 랫 대비 상대적으로 작은 안구 크기를 가진 마우스를 이용하여 안구 내 개발 중인 치료제 전달시 정밀한 실험 조작이 필요하나 현실적으로 시술과 관련한 부작용 발생 가능성이 높아 치료의 평가가 어렵다.In this way, most mouse models of retinal degeneration are developed using mice that are relatively smaller than rats, and precise experimental manipulation is required when delivering therapeutics under development into the eye using mice with relatively smaller eyeballs than rats. However, realistically, treatment evaluation is difficult due to the high possibility of side effects related to the procedure.
한편, 최근 실험 동물로서 랫의 시장 규모는 급격히 증가하고 있으며, 적용 분야는 독성학, 암 종양, 신경계, 면역계 등 다양한 분야에서 활용도가 높아지고 있다. 랫 유전체는 인간의 27억 5천 여개의 유전체 크기와 유사한 특징을 보이며, 이 중에서 2만 5천개의 유전자가 인간과 유사성을 보이는 것으로 보고되었다. 안과용 실험 동물 쥐(랫)와 관련된 통계 결과는 발표된 바 없으나, 신경 질환, 당뇨, 심혈관계, 소화기계, 이식 관련 동물 실험에 활용도가 높아지고 있다. Meanwhile, the market size of rats as laboratory animals is rapidly increasing recently, and their use is increasing in various fields such as toxicology, cancer tumors, nervous system, and immune system. The rat genome shows similar characteristics to the human genome of 2.75 billion genes, and among these, 25,000 genes were reported to show similarity to humans. Although no statistical results have been published regarding rats as ophthalmic laboratory animals, their use in animal experiments related to neurological diseases, diabetes, cardiovascular system, digestive system, and transplantation is increasing.
안과 실험 동물 모델로서 랫은 마우스와 비교하여 상대적으로 큰 안구 크기로 인해 유리체강내 및 망막하 약물 투여 등 다양한 접근 방법이 가능하다. 또한, 상대적으로 고용량의 실험 약물 투여가 가능하여 약물 효능 및 독성 평가에 보다 유용하다. 또한, 랫은 미니 피그, 원숭이 등의 중대 실험동물과 비교해서 실험을 효율적으로 진행하고, 경제적 비용이 감소된다는 점에서 망막색소변성증 동물 모델로서 적합하다. As an animal model for ophthalmological experiments, rats are capable of various approaches, such as intravitreal and subretinal drug administration, due to their relatively large eye size compared to mice. In addition, it is possible to administer relatively high doses of experimental drugs, making it more useful for evaluating drug efficacy and toxicity. In addition, rats are suitable as an animal model for retinitis pigmentosa in that experiments can be conducted more efficiently and economic costs are reduced compared to major laboratory animals such as mini-pigs and monkeys.
망막색소변성증 동물 모델로는 PDE6B 유전자가 결손된 망막색소변성증 동물 모델이 알려져 있다(한국 등록특허 제2177174호). 이 망막색소변성증 동물 모델은 유전자 가위를 사용하여 특이적으로 표적하는 유전자만 제거되어 돌연변이 생성이 안정적이므로 다른 후천적인 요인에 영향을 받지 않고 균일하게 망막색소변성증의 증상을 나타내었다. 하지만, 위 PDE6B 유전자가 결손된 망막색소변성증 동물 모델의 경우 인간과는 다르게 눈 속에 망막색소상피층이 얇기 때문에 망막 시각 기능 평가 및 형광안저검사를 할 수 없어 안과질환 연구시 치료제의 정확한 기전을 알 수 없는 문제점이 있었다. A retinitis pigmentosa animal model lacking the PDE6B gene is known as a retinitis pigmentosa animal model (Korean Patent No. 2177174). This retinitis pigmentosa animal model showed symptoms of retinitis pigmentosa uniformly without being affected by other acquired factors because only the specifically targeted gene was removed using genetic scissors and the generation of mutations was stable. However, in the case of retinitis pigmentosa animal models lacking the above PDE6B gene, unlike humans, the retinal pigment epithelium layer in the eye is thin, so retinal visual function evaluation and fluorescein fundus examination cannot be performed, making it difficult to determine the exact mechanism of the treatment when researching ocular diseases. There was a problem that wasn't there.
롱에반스(Long Evans, 이하 “LE”) 랫은 1915년 알비노 암컷과 야생형 회색 수컷을 교배하여 얻은 실험 동물이며, 털 색은 머리와 어깨 부분은 검은색이고 몸 부분은 흰색이다. 다산종이고, 신경 독성 물질에 민감하여 신경 독성 연구에 쓰이며, 신경 독성 안과 연구에 사용된다. LE 랫은 인간과 유사하게 맥락막, 망막색소상피층이 있고, 시기능이 우수한 것으로 알려져 있다. 망막색소상피층이 있으므로 형광 안저 검사가 가능하며 눈의 형태나 혈관 등을 관찰할 수 있다. The Long Evans (“LE”) rat is a laboratory animal obtained by crossing an albino female and a wild-type gray male in 1915, and its fur color is black on the head and shoulders and white on the body. It is a prolific species and is sensitive to neurotoxic substances, so it is used in neurotoxicity research and in neurotoxic ophthalmic research. LE rats have choroid and retinal pigment epithelial layers similar to humans and are known to have excellent visual function. Because there is a retinal pigment epithelium layer, fluorescent fundus examination is possible and the shape of the eye and blood vessels can be observed.
본 발명자는 망막색소변성증 동물 모델로서 적합한 동물에 대해 연구하였으며, 기존에 개발된 유전자 변형 망막변성 동물 모델인 SD 랫 기반의 PDE6B KO 랫과, 인간과 유사한 안구내 색소를 보유한 정상 롱에반스 랫(Long Evans rat)과 교배를 통해 새로운 망막색소변성증 랫 모델을 개발하였다. 개발된 PDE6B KO 롱에반스 랫 모델은 기존 SD 랫 기반의 동물 모델과 비교하여 시간 경과에 따른 망막색소변성 진행 양상을 보이면서, 인간과 유사한 안구내 색소 특성을 보이므로 안과 질환에 대한 치료제 투여 후 망막 시각 기능 평가 및 형광안저검사 등 영상 검사가 가능하다.The present inventor studied animals suitable as an animal model for retinitis pigmentosa, including PDE6B KO rats based on SD rats, which are previously developed genetically modified retinal degeneration animal models, and normal Long-Evans rats (Long Evans), which have intraocular pigment similar to humans. A new retinitis pigmentosa rat model was developed through crossbreeding with Evans rat). The developed PDE6B KO Long-Evans rat model shows the progression of retinitis pigmentosa over time compared to the existing SD rat-based animal model and shows intraocular pigment characteristics similar to humans, thereby improving retinal vision after administration of treatment for ocular diseases. Imaging tests such as functional evaluation and fluorescein fundus examination are possible.
기존 SD 랫 기반의 동물 모델은 안구 내 색소가 인간과 유사하지 않아 망막 시각 기능 평가 및 형광안저 검사가 어려워 동물 모델을 통한 치료제의 치료 효과 등을 정확히 평가하기 어려웠다. 따라서, 안구 내 색소가 인간과 유사하면서도 시간 경과에 따라 망막색소변성 진행 양상을 보이는 동물 모델의 개발이 필요하다.In the existing SD rat-based animal model, the intraocular pigment is not similar to that of humans, making it difficult to evaluate retinal visual function and fluorescein fundus examination, making it difficult to accurately evaluate the therapeutic effect of the treatment through the animal model. Therefore, there is a need to develop an animal model that shows the progression of retinitis pigmentosa over time while the intraocular pigment is similar to that of humans.
상기 과제를 해결하기 위해, 본 발명의 일 측면은, 하기의 단계를 포함하는 망막변성증 동물 모델의 제조 방법을 제공한다: a) 망막 변성에 관여하는 유전자가 넉아웃(knock-out)된 SD 랫과 야생형 LE 랫을 교배하여 자손 F1을 얻는 단계; b) F1 중에서 이형접합체를 선별하는 단계; c) 선별된 F1 이형접합체와 야생형 LE 랫을 역교배하여 자손 B1을 얻는 단계; d) B1 중에서 털 색이 야생형 LE 랫의 털 색을 나타내고, 안구의 색이 흑색인 개체를 육안으로 1차 선별하는 단계; e) 1차 선별된 B1 중에서 털 색(+/+)인 개체를 2차 선별하고, 2차 선별된 개체 중에서 망막 변성에 관여하는 유전자(+/-)인 개체를 3차 선별하는 단계; f) 3차 선별된 B1 개체를 야생형 LE 랫과 교배하여 자손 B2를 얻는 단계.In order to solve the above problem, one aspect of the present invention provides a method for producing an animal model of retinal degeneration comprising the following steps: a) SD rats in which genes involved in retinal degeneration are knocked out. Obtaining progeny F1 by crossing wild-type LE rats with wild-type LE rats; b) selecting heterozygotes among F1; c) backcrossing the selected F1 heterozygote and wild-type LE rats to obtain progeny B1; d) primary visual selection of individuals among B1 whose fur color represents that of a wild-type LE rat and whose eye color is black; e) secondly selecting individuals with fur color (+/+) among the firstly selected B1, and thirdly selecting individuals with genes involved in retinal degeneration (+/-) among the secondly selected individuals; f) Step of crossing the third selected B1 individual with wild type LE rat to obtain progeny B2.
본 발명의 다른 측면은, 상기 방법으로 제조된 방법으로 제조된 망막변성증 동물 모델을 제공한다.Another aspect of the present invention provides an animal model of retinal degeneration produced by the above method.
본 발명의 또 다른 측면은, i) 망막변성증 동물 모델에 피검 물질을 처리하는 단계; 및 ii) 상기 단계 i)에서 피검 물질이 처리된 망막변성증 동물 모델을 무처리 대조군과 비교하여 망막변성증 증상의 개선 여부를 확인하는 단계를 포함하는, 망막변성증의 치료제 스크리닝 방법을 제공한다.Another aspect of the present invention includes the steps of i) treating a test substance to an animal model of retinal degeneration; and ii) comparing the retinal degeneration animal model treated with the test substance in step i) with an untreated control group to determine whether symptoms of retinal degeneration are improved.
본 발명의 망막변성증 동물 모델의 제조 방법에 의해 제조된 동물 모델은 인간의 눈과 유사하므로, 위 동물 모델을 유전성 망막변성증의 기전 연구에 활용할 수 있다.Since the animal model produced by the method for producing a retinal degeneration animal model of the present invention is similar to the human eye, the above animal model can be used to study the mechanism of hereditary retinal degeneration.
도 1은 이종교배 동물 제작 과정과 PDE6B KO 랫 모체와 이종교배하여 얻은 F1을 나타낸다.
도 2 및 도 3은 이종교배된 랫의 안저 촬영 이미지이다.
도 4 및 도 5는 이종교배된 랫의 빛 간섭 단층 촬영 이미지이다.
도 6은 이종교배된 랫이 6주령일 때의 망막 전위도 검사 결과이다.
도 7은 이종교배된 랫이 10주령일 때의 망막 전위도 검사 결과이다.
도 8은 인간, PDE6B KO 랫 및 야생형 LE 랫의 안구 조직 검사 결과를 나타낸다.
도 9는 이종교배된 랫 및 야생형 LE 랫의 안구 조직 검사 결과를 나타낸다. Figure 1 shows the process of creating crossbreed animals and F1 obtained by crossbreeding with PDE6B KO rat parents.
Figures 2 and 3 are fundus images of crossbred rats.
Figures 4 and 5 are optical coherence tomography images of crossbred rats.
Figure 6 shows the results of an electroretinogram test when a crossbred rat was 6 weeks old.
Figure 7 shows the results of an electroretinogram test when a crossbred rat was 10 weeks old.
Figure 8 shows eye tissue examination results from humans, PDE6B KO rats, and wild-type LE rats.
Figure 9 shows the results of eye tissue examination of crossbred rats and wild-type LE rats.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
하기 “1.망막변성증 동물 모델의 제조 방법”에서는 망막변성증 동물 모델의 제조 방법과 이 방법에 의해 제조된 동물 모델에 대해서 설명하고, “2. 망막변성증의 치료제 스크리닝 방법”에서는 위 망막변성증 동물 모델의 치료제 스크리닝에 대한 용도에 대해서 설명한다.In “ 1. Method for producing a retinal degeneration animal model ” below, the method for producing a retinal degeneration animal model and the animal model produced by this method are described, and in “ 2. Screening method for a therapeutic agent for retinal degeneration ”, the above retinal degeneration animal model is described. The use for therapeutic screening is explained.
1.One. 망막변성증 동물 모델의 제조 방법Method for producing a retinal degeneration animal model
본 발명에서 사용되는 용어, “동물 모델”이란 사람의 질병과 아주 유사한 형태의 질병을 가진 동물을 말한다. 사람의 질병 연구에 있어 질환 모델 동물이 의미를 갖는 것은 사람과 동물들 간의 생리적 또는 유전적인 유사성에 의한다. 질병 연구에 있어 질환 모델 동물은 질병의 다양한 원인과 발병 과정 및 진단에 대한 연구용 재료를 제공해주고, 질환 모델 동물의 연구를 통해 질병에 관련된 유전자들을 알아내며, 유전자들 간의 상호작용을 이해할 수 있게 하고, 개발된 신약 후보 물질의 실제 효능 및 독성 검사를 통해 실용화 가능성의 여부를 판단하는 기초 자료를 얻을 수 있다. As used in the present invention, the term “animal model” refers to an animal that has a disease that is very similar to a human disease. The significance of disease model animals in the study of human diseases is due to the physiological or genetic similarities between humans and animals. In disease research, disease model animals provide materials for research into various causes, pathogenesis, and diagnosis of diseases, and through research on disease model animals, genes related to diseases can be identified, and interactions between genes can be understood. , basic data to determine whether commercialization is possible can be obtained through actual efficacy and toxicity tests of developed new drug candidates.
본 발명은 하기의 단계를 포함하는 망막변성증 동물 모델의 제조 방법을 제공한다: a) 망막 변성에 관여하는 유전자가 넉아웃(knock-out)된 SD 랫과 야생형 LE 랫을 교배하여 자손 F1을 얻는 단계; b) F1 중에서 이형접합체를 선별하는 단계; c) 선별된 F1 이형접합체와 야생형 LE 랫을 역교배하여 자손 B1을 얻는 단계; d) B1 중에서 털 색이 야생형 LE 랫의 털 색을 나타내고, 안구의 색이 흑색인 개체를 육안으로 1차 선별하는 단계; e) 1차 선별된 B1 중에서 털 색(+/+)인 개체를 2차 선별하고, 2차 선별된 개체 중에서 망막 변성에 관여하는 유전자(+/-)인 개체를 3차 선별하는 단계; f) 3차 선별된 B1 개체를 야생형 LE 랫과 교배하여 자손 B2를 얻는 단계.The present invention provides a method for producing an animal model of retinal degeneration comprising the following steps: a) crossing SD rats in which genes involved in retinal degeneration have been knocked out and wild-type LE rats to obtain offspring F1. step; b) selecting heterozygotes among F1; c) backcrossing the selected F1 heterozygote and wild-type LE rats to obtain progeny B1; d) primary visual selection of individuals among B1 whose fur color represents that of a wild-type LE rat and whose eye color is black; e) secondly selecting individuals with fur color (+/+) among the firstly selected B1, and thirdly selecting individuals with genes involved in retinal degeneration (+/-) among the secondly selected individuals; f) Step of crossing the third selected B1 individual with wild type LE rat to obtain progeny B2.
상기 망막변성증은 망막색소변성증일 수 있다. 본 발명의 일 실시예에서는 PDE6B KO SD 랫과 야생형 LE 랫을 교배함으로써 망막 변성 양상을 보이면서 안구 조직 내에 망막색소상피층과 맥락막 색소층을 가지는 이종교배된 랫을 얻었다. 이종교배된 랫은 보다 인간과 유사한 해부학적 특성을 가지면서도 망막변성증 증상을 나타내므로 망막변성증, 나아가 망막색소변성증의 연구에 유용하게 사용될 수 있다(실시예 1 및 실시예 3 참조).The retinal degeneration may be retinitis pigmentosa. In one example of the present invention, by crossing PDE6B KO SD rats and wild-type LE rats, crossbred rats showing retinal degeneration and having a retinal pigment epithelial layer and a choroidal pigment layer in the eye tissue were obtained. Since crossbred rats have anatomical characteristics more similar to humans and show symptoms of retinal degeneration, they can be usefully used in the study of retinal degeneration and even retinitis pigmentosa (see Examples 1 and 3).
상기 SD(Sprague-Dawley) 랫은 색소가 없어서 흰색 털에 적색 눈을 가진다. 생물 검정용, 영양 시험용으로 주로 쓰이고 있다. The SD (Sprague-Dawley) rat has no pigment and has white fur and red eyes. It is mainly used for bioassays and nutritional tests.
상기 야생형 LE(Long-Evans) 랫은 인간과 유사하게 맥락막, 망막색소상피층이 있고, 시기능이 우수한 것으로 알려져 있다. 야생형 LE 랫은 망막변성증 양상을 나타내지 않기 때문에 망막변성증 치료제를 처리하여도 망막변성증이 개선되는지 여부는 확인할 수 없다. 따라서, 야생형 LE 랫을 망막변성증 동물 모델로 사용하기 위해서는 특정 유전자의 결손이나, 망막변성증을 유도하는 물질의 처리 등이 필요하다. 본 발명의 일 실시예에서는 PDE6B KO SD 랫과 야생형 LE 랫을 교배함으로써 망막 변성 양상을 보이면서 안구 조직 내에 망막색소상피층과 맥락막 색소층을 가지는 이종교배된 랫을 얻었다. 이종교배된 랫은 인간과 유사한 해부학적 특성을 가지면서 망막변성증 양상을 나타내므로 망막변성증, 나아가 망막색소변성증의 연구에 유용하게 사용될 수 있다(실시예 1 및 실시예 3 참조).The wild-type LE (Long-Evans) rat has choroid and retinal pigment epithelial layers similar to humans, and is known to have excellent visual function. Because wild-type LE rats do not show signs of retinal degeneration, it cannot be confirmed whether treatment with a treatment for retinal degeneration improves retinal degeneration. Therefore, in order to use wild-type LE rats as an animal model for retinal degeneration, deletion of a specific gene or treatment with a substance that induces retinal degeneration is required. In one example of the present invention, by crossing PDE6B KO SD rats and wild-type LE rats, crossbred rats showing retinal degeneration and having a retinal pigment epithelial layer and a choroidal pigment layer in the eye tissue were obtained. Since crossbred rats have anatomical characteristics similar to humans and show signs of retinal degeneration, they can be usefully used in the study of retinal degeneration and even retinitis pigmentosa (see Examples 1 and 3).
망막 변성에 관여하는 유전자는 PDE6B일 수 있고, 망막 변성에 관여하는 유전자라면 본 발명에 제한없이 포함될 수 있다. 예를 들어, CA4, CRX, FXCN2, GUCA1B, IMPDH1, NR2E3, NRL, PRPF3, PRPF3, PRPF3, PRPF31, PRPH2, RDH12, RHO, ROM1, RP1, RP9, SEMA4A, TOPORS, ABCA4, CERKL, CNGA1, CRB1, EYS, IDH3B, LRAT, MERTK, NR2E3, NRL, PDE6A,PRCD, PROM1, RGR, RHO, RLBP1, RP1, RPE65, SAG, TULP1, USH2A, RP2 및 RPGR로 이루어진 군으로부터 선택되는 어느 하나 이상의 유전자일 수 있다. The gene involved in retinal degeneration may be PDE6B, and any gene involved in retinal degeneration may be included in the present invention without limitation. For example, CA4, CRX, FXCN2, GUCA1B, IMPDH1, NR2E3, NRL, PRPF3, PRPF3, PRPF3, PRPF31, PRPH2, RDH12, RHO, ROM1, RP1, RP9, SEMA4A, TOPORS, ABCA4, CERKL, CNGA1, CRB1, It may be any one or more genes selected from the group consisting of EYS, IDH3B, LRAT, MERTK, NR2E3, NRL, PDE6A, PRCD, PROM1, RGR, RHO, RLBP1, RP1, RPE65, SAG, TULP1, USH2A, RP2 and RPGR. .
또한, 본 발명의 망막 변성에 관여하는 유전자는 망막색소변성증을 유도하는 유전자를 포함한다. 망막색소변성증을 유도하는 유전자는 하기와 같다: ABCA4, AIPL1, ALMS1, ARL6, ATF6, BBS2, BEST1, C2orf71, CA4, CACNA1F, CACNA2D4, CDH23, CDHR1, CERKL, CLRN1, CNGA1, CNGB1, CNNM4, CRB1, CRX, DFNB31, DHDDS, EYS, FAM161A, FLVCR1, FSCN2, GUCA1A, GUCA1B, GUCY2D, HARS, HGSNAT, IDH3B, IFT140, IFT172, IL1A, IL1B, IMPDH1, IMPG2, KCNV2, KLHL7, LRAT, MAK, MFRP, MYO7A, NPHP4, NR2E3, NRL, OFD1, PANK2, PCDH15, PDE6A, PDE6B, PDE6C, PDE6G, PDZD7, PITPNM3, PROM1, PRPF3, PRPF31, PRPF6, PRPF8, PRPH2, RBP3, RDH12, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP2, RPE65, RPGR, RPGRIP1, RRM2B,, SAG, SEMA4A, SNRNP200, SPATA7, TOPORS, TTC8, TTPA, TULP1, UNC119, USH1G, USH2A, ZNF408, ZNF513. 본 발명의 일 실시예에서는 망막색소변성증 유발 유전자인 PDE6B가 결손된 PDE6B KO SD 랫과 야생형 LE 랫을 교배함으로써 망막 변성 양상을 보이면서 안구 조직 내에 망막색소상피층과 맥락막 색소층을 가지는 이종교배된 랫을 얻었다. 이종교배된 랫은 인간과 유사한 해부학적 특성을 가지면서 망막변성증 양상을 나타내므로 망막변성증, 나아가 망막색소변성증의 연구에 유용하게 사용될 수 있다(실시예 1 및 실시예 3 참조).Additionally, genes involved in retinal degeneration of the present invention include genes that induce retinitis pigmentosa. Genes that induce retinitis pigmentosa are as follows: ABCA4, AIPL1, ALMS1, ARL6, ATF6, BBS2, BEST1, C2orf71, CA4, CACNA1F, CACNA2D4, CDH23, CDHR1, CERKL, CLRN1, CNGA1, CNGB1, CNNM4, CRB1, CRX, DFNB31, DHDDS, EYS, FAM161A, FLVCR1, FSCN2, GUCA1A, GUCA1B, GUCY2D, HARS, HGSNAT, IDH3B, IFT140, IFT172, IL1A, IL1B, IMPDH1, IMPG2, KCNV2, KLHL7, LRAT, MAK, MFRP, MYO7A, NPHP4, NR2E3, NRL, OFD1, PANK2, PCDH15, PDE6A, PDE6B, PDE6C, PDE6G, PDZD7, PITPNM3, PROM1, PRPF3, PRPF31, PRPF6, PRPF8, PRPH2, RBP3, RDH12, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP2, RPE65, RPGR, RPGRIP1, RRM2B,, SAG, SEMA4A, SNRNP200, SPATA7, TOPORS, TTC8, TTPA, TULP1, UNC119, USH1G, USH2A, ZNF408, ZNF513. In one embodiment of the present invention, a crossbred rat showing retinal degeneration and having a retinal pigment epithelial layer and a choroidal pigment layer in the eye tissue was produced by crossing a PDE6B KO SD rat lacking PDE6B, a gene causing retinitis pigmentosa, with a wild-type LE rat. got it Since crossbred rats have anatomical characteristics similar to humans and show signs of retinal degeneration, they can be usefully used in the study of retinal degeneration and even retinitis pigmentosa (see Examples 1 and 3).
망막 변성은 망막색소변성, 혈관무늬병증, 드루젠 및 황반 변성으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 또한, 다양한 망막 질환이 망막 변성에 포함된다. 예를 들어, 망막 이영양증, 색소성 망막염, 황반 변성, 레버 선천적 흑암시, 콘-로드 이영양증, 눈의 신생혈관 질환, 맥락막 변성, 맥락막 경화증, 범맥락막 위축, 및 대사 장애, 예컨대 슬라이 증후군(베타-글루코로니다아제 유전자의 결손에 기인한, MPS VII) 및 망막 위축(gyrate atrophy)(오르니틴-델타-아미노트랜스퍼라아제 유전자, OAT의 결손에 기인함), 망막 박리 또는 외상 및 망막 병증(유전성, 수술에 의하여 유도, 외상, 독성 화합물 또는 제제, 또는 광유도에 의한 어떤 것이든 포함)이 본 발명의 망막 변성에 포함될 수 있다. 나아가 망막의 손상 또는 발달 저해로 인해 유발될 수 있는 질환으로서 본 기술 분야의 통상의 기술자에 의해 인정되는 범위라면 제한없이 적용할 수 있다.Retinal degeneration may be any one or more selected from the group consisting of retinitis pigmentosa, angiopathy, drusen, and macular degeneration. Additionally, various retinal diseases are included in retinal degeneration. For example, retinal dystrophy, retinitis pigmentosa, macular degeneration, Leber congenital amaurosis, Con-Rod dystrophy, neovascular disease of the eye, choroidal degeneration, choroidal sclerosis, panchoroidal atrophy, and metabolic disorders such as Sly syndrome (beta- MPS VII) and gyrate atrophy (due to defects in the ornithine-delta-aminotransferase gene, OAT), retinal detachment or trauma and retinopathy (hereditary , surgically induced, trauma, toxic compounds or agents, or light-induced) may be included in the retinal degeneration of the present invention. Furthermore, it can be applied without limitation as long as it is a disease that can be caused by damage or developmental inhibition of the retina and is within the scope recognized by those skilled in the art.
망막 변성에 관여하는 유전자의 결손은 본 기술 분야에 공지된 유전자 교정 기술에 의해 수행될 수 있다. 구체적으로 CRISPR 유전자 가위 기술을 이용해 망막 변성에 관여하는 유전자가 넉아웃된 랫을 제조할 수 있다. 예를 들어, 한국 등록특허 제2177174호에 개시된 PDE6B KO 랫의 제조 방법을 사용할 수 있다. Deletion of genes involved in retinal degeneration can be performed by gene editing techniques known in the art. Specifically, using CRISPR gene scissors technology, it is possible to produce rats in which genes involved in retinal degeneration are knocked out. For example, the method for producing PDE6B KO rats disclosed in Korean Patent No. 2177174 can be used.
b) 단계에서 F1 중에서 이형접합체 선별은 육안으로 수행되며 털 색이 야생형 LE 랫의 털 색인 개체를 선별하는 것일 수 있다. In step b), heterozygous selection among F1 is performed visually and may be to select individuals whose fur color is that of wild-type LE rats.
e) 단계에서 1차 선별된 B1 중에서 털 색(+/+)인 개체의 선별은 1차 선별된 B1 랫(♀)과 SD(알비노) 랫(♂)을 교배하여 야생형 LE 랫과 같은 털 색을 가지는 자손만을 생산하는 B1 개체 모체를 선별하는 것일 수 있다. 1차 선별된 B1 중에서 털 색(+/+)인 개체(♀)는 SD(알비노) 랫(♂)와 교배시 야생형 LE 랫과 같은 털 색을 가진 자손만을 생산하지만, 1차 선별된 B1 중에서 털 색(+/-)인 개체(♀)는 SD(알비노) 랫(♂)와 교배시 야생형 LE 랫과 같은 털 색을 가진 자손, 또는 알비노인 자손을 생산한다. 따라서, 1차 선별된 B1의 유전자형의 동형접합 여부를 1차 선별된 B1과 SD(알비노) 랫을 교배하여 자손의 털 색을 확인함으로써 알 수 있다.In step e), individuals with fur color (+/+) among the first selected B1 are selected by crossing the first selected B1 rat (♀) with SD (albino) rat (♂) to obtain the same fur color as the wild-type LE rat. This may be selecting B1 individual mothers that produce only offspring with . Among the B1s selected first, individuals with fur color (♀) produce only offspring with the same fur color as wild-type LE rats when crossed with SD (albino) rats (♂); however, among the B1s selected firstly, When an individual (♀) with fur color (+/-) is crossed with an SD (albino) rat (♂), it produces offspring with the same fur color as a wild-type LE rat, or offspring that are albino. Therefore, whether the genotype of the first selected B1 is homozygous can be determined by crossing the first selected B1 and SD (albino) rats and checking the fur color of the offspring.
e) 단계에서 망막 변성에 관여하는 유전자(+/-)인 개체의 선별은 망막 변성에 관여하는 유전자 검출용 프라이머 세트를 이용하는 것일 수 있다. 상기 망막 변성에 관여하는 유전자 검출용 프라이머는 망막 변성에 관여하는 유전자를 타겟시퀀스로 하여 Primer3 프로그램과 같은 프라이머 디자인 프로그램을 이용하여 제작될 수 있다. In step e), selection of individuals with genes involved in retinal degeneration (+/-) may be done using a primer set for detecting genes involved in retinal degeneration. Primers for detecting genes involved in retinal degeneration can be produced using a primer design program such as the Primer3 program using genes involved in retinal degeneration as target sequences.
상기 망막 변성에 관여하는 유전자가, PDE6B인 경우 PDE6B 유전자 검출용 프라이머 세트는 서열번호 1의 염기 서열로 이루어지는 정방향 프라이머; 및 서열번호 2의 염기 서열로 이루어지는 역방향 프라이머를 포함할 수 있다.When the gene involved in retinal degeneration is PDE6B, the primer set for detecting the PDE6B gene includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1; And it may include a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.
상기 유전자는 각 유전자에 상보적으로 결합하는 염기서열로 이루어진 프라이머 세트를 이용하여 PCR을 수행하여 검출될 수 있다.The gene can be detected by performing PCR using a primer set consisting of a base sequence that binds complementary to each gene.
본 발명의 용어 “프라이머”는 적합한 온도 및 완충액 내에서 적합한 조건(즉, 4종의 다른 뉴클레오타이드 트리포스페이트 및 중합반응 효소) 하에서 주형-지시 DNA 합성의 개시점으로 작용할 수 있는 단일-가닥 올리고뉴클레오타이드를 의미한다. The term “primer” of the present invention refers to a single-stranded oligonucleotide that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleotide triphosphates and polymerization enzymes) at a suitable temperature and buffer. it means.
본 발명의 용어 “역전사 중합효소 연쇄반응(RT-PCR)” 기법은 중합효소 연쇄 반응 기법(Polymerase Chain Reaction, PCR) 이용하는 분자 기법 중 하나이다. RNA가 역전사 효소에 의해 역전사되어 cDNA를 만들어지고, 이 cDNA를 주형틀이 되어 중합효소 연쇄반응(Polymerase Chain Reaction, PCR)이 진행된다. PCR 산물의 양을 측정함으로써, cDNA와 mRNA의 양을 간접적으로 알 수 있다.The term “reverse transcription polymerase chain reaction (RT-PCR)” in the present invention is one of the molecular techniques using the polymerase chain reaction (Polymerase Chain Reaction) technique. RNA is reverse transcribed by reverse transcriptase to create cDNA, and this cDNA is used as a template for polymerase chain reaction (PCR). By measuring the amount of PCR product, the amount of cDNA and mRNA can be indirectly determined.
c) 단계 내지 f) 단계는 반복되며, B3 이상의 자손까지, B4 이상의 자손까지, B5 이상의 자손까지, B6 이상의 자손까지, B7 이상의 자손까지, B8 이상의 자손까지, B9 이상의 자손까지, B10 이상의 자손까지, B11 이상의 자손까지, 또는 그 이상의 자손까지 진행되는 것일 수 있다. 일정한 표현형을 가지는 개체를 얻기 위해서 수차례 반복될 수 있다. Steps c) to f) are repeated, up to progeny B3 and higher, up to progeny B4 and higher, up to progeny B5 and higher, up to progeny B6 and higher, up to progeny B7 and higher, up to progeny B8 and higher, up to progeny B9 and higher, up to progeny B10 and higher. , it may progress to B11 or higher offspring, or even higher offspring. It can be repeated several times to obtain individuals with a certain phenotype.
상기 망막변성증 동물 모델의 제조 방법에 의해 얻어진 망막변성증 동물 모델의 안구 조직 내에는 망막색소상피층과 맥락막 색소층이 존재할 수 있다.A retinal pigment epithelial layer and a choroidal pigment layer may be present in the eye tissue of the retinal degeneration animal model obtained by the method for producing the retinal degeneration animal model.
또한 본 발명은 상기 방법으로 제조된 망막변성증 동물 모델을 제공한다.The present invention also provides a retinal degeneration animal model prepared by the above method.
2.2. 망막변성증의 치료제 스크리닝 방법Screening method for treatments for retinal degeneration
본 발명은 하기 단계를 포함하는 망막변성증의 치료제 스크리닝 방법을 제공한다: i) 상기 망막변성증 동물 모델의 제조 방법에 의해 제조된 망막변성증 동물 모델에 피검 물질을 처리하는 단계; 및 ii) 단계 i)에서 피검 물질이 처리된 망막변성증 동물 모델을 무처리 대조군과 비교하여 망막변성증 증상의 개선 여부를 확인하는 단계.The present invention provides a screening method for a therapeutic agent for retinal degeneration, comprising the following steps: i) treating a test substance to a retinal degeneration animal model prepared by the method for producing a retinal degeneration animal model; and ii) comparing the retinal degeneration animal model treated with the test substance in step i) with the untreated control group to determine whether symptoms of retinal degeneration are improved.
상기 단계 ii)의 망막변성증 증상의 개선은, 망막 혈관 형태의 개선, 망막 단층 두께의 증가, 망막 전위도의 증가 및 망막 조직 내 세포수 증가로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있고, 망막변성으로 인한 증상이라면 제한없이 본 발명에 포함된다. The improvement in the symptoms of retinal degeneration in step ii) may be any one or more selected from the group consisting of improvement in retinal vascular morphology, increase in retinal monolayer thickness, increase in electroretinogram, and increase in the number of cells in retinal tissue, and retinal degeneration Symptoms caused by are included in the present invention without limitation.
상기 피검 물질은 화합물, 추출물, 유전자 치료제 및 세포 치료제로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. The test substance may be any one or more selected from the group consisting of compounds, extracts, gene therapy agents, and cell therapy agents.
이하, 본 발명을 구체적으로 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to these only.
실시예 1. PDE6B 결손 SD 랫과 야생형 롱에반스 랫을 교배하여 정상 망막 색소를 가지는 이종교배 동물 모델 제조Example 1. Preparation of a crossbreed animal model with normal retinal pigment by crossing PDE6B-deficient SD rats with wild-type Long-Evans rats.
1-1.1-1. 교배 방법breeding method
기존에 개발된 유전자 변형 망막변성 동물 모델인 SD 랫 기반의 PDE6B KO(이하, “PDE6B KO SD”) 랫과 망막 색소가 정상인 야생형 롱에반스(Long-Evans, 이하 “LE”) 랫을 교배하여 망막 색소가 정상인 PDE6B KO 랫 제작을 시도하였다. PDE6B KO SD 랫은 한국 등록특허 제2177174호에 개시되어 있는 PDE6B 유전자가 결손된 망막 변성 동물 모델의 제조 방법으로 제조되었다. 개체 변화 없이 일정한 표현형을 내기 위해서 역교배(backcross) 과정을 거쳐 백그라운드를 제작하였다. 동물 모델 제작 과정은 하기와 같다(도 1 참조). The retina was developed by crossing PDE6B KO (hereinafter “PDE6B KO SD”) rats based on SD rats, a previously developed animal model for genetically modified retinal degeneration, with wild-type Long-Evans (hereinafter “LE”) rats with normal retinal pigment. An attempt was made to produce PDE6B KO rats with normal pigmentation. PDE6B KO SD rats were prepared using the method for producing a retinal degeneration animal model with a defect in the PDE6B gene disclosed in Korean Patent No. 2177174. In order to produce a consistent phenotype without individual change, a background was created through a backcross process. The animal model production process is as follows (see Figure 1).
a) PDE6B KO SD 랫(♀)과 야생형 LE 랫(♂)을 교배하여 자손 F1을 얻는다(도 1 참조). a) Crossbreeding PDE6B KO SD rat (♀) with wild type LE rat (♂) to obtain offspring F1 (see Figure 1).
b) F1 중에서 이형접합체(heterozygote, 이하 “hetero”)를 선별한다. b) Select heterozygotes (hereinafter “hetero”) among F1.
c) 선별된 F1(hetero)(♀)을 야생형 LE 랫(♂)과 역교배하여 자손 B1을 얻는다. c) Backcross the selected F1 (hetero) (♀) with the wild type LE rat (♂) to obtain progeny B1.
d) B1 중에서 털 색이 야생형 LE 랫의 털 색을 나타내고, 안구의 색이 흑색인 개체(♀)를 육안으로 1차 선별한다. d) Among B1, individuals whose fur color represents that of a wild-type LE rat and whose eye color is black (♀) are initially selected with the naked eye.
e) 단계는 1차 선별된 B1 중에서 털 색(+/+)이고, 망막 변성에 관여하는 유전자인 PDE6B (+/-)인 개체를 선별하는 단계이며, e-1) 단계에서 털 색(+/+)인 개체를 2차 선별하고, e-2) 단계를 통해 2차 선별된 개체 중에서 PDE6B (+/-)인 개체를 3차 선별한다. Step e) is the step of selecting individuals with fur color (+/+) and PDE6B (+/-), a gene involved in retinal degeneration, among the first selected B1, and in step e-1), hair color (+) /+) individuals are secondarily screened, and PDE6B (+/-) individuals are thirdly selected among the individuals secondarily selected through step e-2).
e-1) 1차 선별된 B1 중에서, B1(♀)을 SD(알비노) 랫(♂)과 교배하여 야생형 LE 랫과 같은 털 색을 가지는 자손만을 생산하는 B1 개체 모체, 즉 털 색(+/+)인 B1을 2차 선별한다. e-1) Among the first selected B1, B1 (♀) was crossed with SD (albino) rat (♂) to produce only offspring with the same fur color as wild-type LE rat, B1 individual parent, that is, fur color (+/ B1, which is +), is subjected to secondary selection.
구체적으로, 랫에서 털 색은 유색이 우성이고, 무색(알비노)이 열성이다. B1이 털 색에 대해서 동형접합체(+/+)일 경우 SD(알비노) 랫과 교배하면 모든 자손이 LE 털 색을 갖는다. 반면, B1이 털 색에 대해 이형접합체(+/-)일 경우 자손의 털 색이 LE 털 색일 수도 있고, 알비노일 수도 있다. 따라서, B1의 털 색에 대한 유전자형을 확인하기 위해서, B1(♀)과 SD(알비노) 랫(♂)의 교배를 통한 자손의 털 색 확인 과정이 필요하다.Specifically, in rats, colored hair is dominant and colorless (albino) is recessive. If B1 is homozygous (+/+) for fur color and is bred with an SD (albino) rat, all offspring will have LE fur color. On the other hand, if B1 is heterozygous (+/-) for fur color, the offspring may have LE fur color or may be albino. Therefore, in order to confirm the genotype for the fur color of B1, it is necessary to confirm the fur color of the offspring by crossing B1 (♀) and SD (albino) rats (♂).
e-2) 2차 선별된 B1 개체(♀)를 하기 실시예 1-2의 유전자형 판별법을 이용하여 PDE6B 유전자가 이형접합체(+/-)인 개체를 3차 선별한다. e-2) The secondly selected B1 individual (♀) is subjected to a third selection of individuals heterozygous for the PDE6B gene (+/-) using the genotype determination method in Example 1-2 below.
f) 3차 선별된 B1 개체(♀)를 다시 야생형 LE 랫(♂)과 교배한다. f) The third selected B1 individual (♀) is crossed again with the wild type LE rat (♂).
g) c) 내지 f)를 반복하여 B5 이상의 자손까지 진행한다. g) Repeat c) to f) to proceed to B5 or higher offspring.
PDE6B KO SD 랫과 야생형 LE 랫을 교배하여 얻어진 망막 색소가 정상인 PDE6B KO 랫은 육안으로 확인시 털 색이 머리와 어깨 부분은 까만색이고, 몸 부분은 하얀색을 나타내며, 안구는 야생형 LE 랫과 같은 흑색을 나타낸다. PDE6B KO 랫과 야생형 LE 랫을 교배하여 얻어진 망막 색소가 정상인 PDE6B KO 랫은 이하에서 '이종교배된 동물 모델', 또는 '이종교배된 랫'으로 기재한다.PDE6B KO rats with normal retinal pigment, obtained by crossing PDE6B KO SD rats and wild-type LE rats, have black hair on the head and shoulders, white on the body, and black eyes like wild-type LE rats. represents. PDE6B KO rats with normal retinal pigment obtained by crossing PDE6B KO rats with wild-type LE rats are hereinafter referred to as 'crossbred animal models' or 'crossbred rats'.
1-2.1-2. 유전자형 판별(genotyping)Genotyping
실시예 1-1에서 얻은 이종교배된 랫이 1주령일 때 랫의 꼬리를 잘라내어(tail cut) gDNA를 추출해 내었다. 추출한 gDNA를 주형으로 하여 하기 표 1의 프라이머를 사용하고, 표 2의 PCR 조건으로 PCR을 수행하였다. PCR 산물은 2.0% 아가로스 젤에 로딩하여 확인하였다.When the crossbred rat obtained in Example 1-1 was 1 week old, its tail was cut and gDNA was extracted. Using the extracted gDNA as a template, the primers in Table 1 below were used, and PCR was performed under the PCR conditions in Table 2. PCR products were confirmed by loading on a 2.0% agarose gel.
유전자PDE6B
gene
68℃95℃56℃
68℃
15초
1분15 seconds
15 seconds
1 min
PCR 산물을 확인한 결과, PDE6B KO SD 랫과 야생형 LE 랫을 교배하여 얻어진 자손 중에서 육안으로 확인시 털 색이 머리와 어깨 부분은 까만색이고, 몸 부분은 하얀색을 나타내며, 안구는 야생형 LE 랫과 같은 흑색을 나타내는 개체는 털 색 유전자가 야생형 동형접합체(+/+) wildtype homozygote)였고, PDE6B 유전자가 이형접합체(+/- heterozygote, 한쪽 염색체만 KO)인 것으로 확인되었다. As a result of checking the PCR product, among the offspring obtained by crossing a PDE6B KO SD rat and a wild-type LE rat, when confirmed with the naked eye, the hair color of the head and shoulders is black, the body is white, and the eyes are black like the wild-type LE rat. The individual showing was confirmed to be wild-type homozygote (+/+) wildtype homozygote for the fur color gene and heterozygote (+/- heterozygote (KO only one chromosome) for the PDE6B gene.
실시예 2. 이종교배된 랫의 망막 변성 여부 확인Example 2. Confirmation of retinal degeneration in crossbred rats
2-1. 안저 촬영2-1. fundus photography
본 발명에서 제작한 이종교배된 랫을 망막변성증의 동물 모델로서 사용할 수 있는지 확인하기 위해, 망막의 전반적인 소견을 확인하였다.In order to confirm whether the crossbred rat produced in the present invention can be used as an animal model for retinal degeneration, the overall findings of the retina were confirmed.
상기 실시예 1-1에서 제조된 이종교배된 랫이 3주령, 4주령, 5주령, 6주령, 8주령 9주령일 때 안저 촬영을 시행하였다. 랫의 안구를 점안 마취제로 마취하고, 5 ㎎/㎖ 트로피카마이드(tropicamide) 및 5 ㎎/ml 페닐프린 HCl(phenylephrine HCL)을 점안하여 동공의 산동을 유도하였다. 안저 촬영 중 각막의 건조를 방지하기 위해 안과용 인공 누액 연고를 사용하였다. 망막 촬영은 Micron IV 안저 카메라를 사용하였다(Phoenix Research Labs, Inc., Pleasanton, CA, USA). 촬영한 모든 안저 영상은 Micron IV software(StreamPix; NorPix, Inc. Montreal, Quebec, Canada)를 이용하여 저장하고 데이터 처리하였다. Fundus photography was performed when the crossbred rats prepared in Example 1-1 were 3 weeks old, 4 weeks old, 5 weeks old, 6 weeks old, 8 weeks old, and 9 weeks old. The rat's eye was anesthetized with an eye anesthetic, and mydriasis was induced by instilling 5 mg/ml tropicamide and 5 mg/ml phenylephrine HCL. To prevent drying of the cornea during fundus photography, ophthalmic artificial tear ointment was used. Retinal imaging was performed using a Micron IV fundus camera (Phoenix Research Labs, Inc., Pleasanton, CA, USA). All fundus images taken were stored and data processed using Micron IV software (StreamPix; NorPix, Inc. Montreal, Quebec, Canada).
그 결과, 이종교배된 랫은 안저 촬영 소견상 시신경을 중심으로 하여 방사형으로 주행하는 망막 혈관의 형태가 불규칙하며, 망막 혈과 두께가 감소되는 양상을 나타내었다(도 2 및 도 3 참조).As a result, the crossbred rat showed an irregular shape of the retinal blood vessels running radially around the optic nerve, and a decrease in retinal blood volume and thickness, as shown in fundus photography (see Figures 2 and 3).
2-2. 빛 간섭 단층 촬영2-2. Optical coherence tomography
본 발명에서 제작한 이종교배된 랫을 망막변성증의 동물 모델로서 사용할 수 있는지 확인하기 위해, 스펙트럼 빛 간섭 단층 촬영(Spectral-domain Optical coherence tomography, SD-OCT)을 통해 망막의 단층 소견을 확인하였다.In order to confirm whether the crossbred rat produced in the present invention can be used as an animal model for retinal degeneration, retinal tomographic findings were confirmed through spectral-domain optical coherence tomography (SD-OCT).
상기 실시예 1-1에서 제조된 이종교배된 랫이 3주령, 4주령, 5주령, 6주령, 8주령 9주령일 때 빛 간섭 단층 촬영(Phoenix Research Labs)을 수행하였다. 6 번 반복하여 망막 단층을 스캔하고, 그 값을 평균화하여 영상을 획득하였다. 획득된 영상은 tagged image file(.tif) 형식으로 출력하여, 망막의 두께 및 망막색소상피(retinal pigment epithelium), 망막의 외핵층(outer nuclear layer), 외망상층(outer plexiform layer), 내핵층(inner nuclear layer) 및 내망막층(inner plexiform layer) 등의 양상을 비교하였다. Optical coherence tomography (Phoenix Research Labs) was performed on the crossbred rats prepared in Example 1-1 when they were 3 weeks old, 4 weeks old, 5 weeks old, 6 weeks old, 8 weeks old, and 9 weeks old. The retinal tomography was scanned repeatedly 6 times, and the values were averaged to obtain an image. The acquired images are output in tagged image file (.tif) format, and the thickness of the retina, retinal pigment epithelium, outer nuclear layer, outer plexiform layer, and inner nuclear layer of the retina are displayed. Aspects such as (inner nuclear layer) and inner retina layer (inner plexiform layer) were compared.
그 결과, 이종교배된 랫에서 망막 전반의 두께가 감소되는 양상을 보였으며 시세포층의 손상이 확인되었다(도 4 및 도 5 참조). As a result, the thickness of the overall retina was decreased in the crossbred rats, and damage to the photoreceptor cell layer was confirmed (see Figures 4 and 5).
2-3. 망막 전위도 검사2-3. electroretinogram
본 발명에서 제작한 이종교배된 랫을 망막변성증의 동물 모델로서 사용할 수 있는지 확인하기 위해, 망막 전위도 검사를 수행하였다.To confirm whether the crossbred rat produced in the present invention can be used as an animal model for retinal degeneration, an electroretinogram test was performed.
상기 실시예 1-1에서 제조된 이종교배된 랫이 6주령, 10주령일 때, 망막 전위도 검사를 수행하였다. 정상 대조군은 야생형 LE 랫으로 하였다. 망막전위도 검사 전 랫은 각각 12 시간 이상 암순응을 유도하였고, 조도(λ) 600 nm 미만의 어두운 환경에서 망막 전위도 측정을 준비하였다. 준비 후, 각각의 랫에 Zoletil®(Tiletamine 125 mg/ml and Zolazepam 125 mg/ml, Virbac, France)을 0.01 ml 씩 복강내 주사하여 마취하였다. 또한, 미드린-피 점안제®(phenylephrine hydro-chloride 5㎎/ml 및 tropicamide 5 ㎎/ml, Santen, Japan)를 점안하여 동공을 산동시켰다. 또한, 안구 마취를 위해 Alcaine®(proparacaine hydrochloride0.5 %, Alcon Laboratories Inc.)을 점안하였다. 마취된 랫은 실험동물용 지지대에 안정적으로 고정시켜, 재현성 높은 전기생리학적 검사를 위한 체위를 유지하였다.Electroretinogram tests were performed when the crossbred rats prepared in Example 1-1 were 6 and 10 weeks old. The normal control group was wild-type LE rats. Before the electroretinogram test, each rat was induced to dark adapt for more than 12 hours, and electroretinogram measurement was prepared in a dark environment with an illuminance (λ) of less than 600 nm. After preparation, each rat was anesthetized by intraperitoneally injecting 0.01 ml of Zoletil® (Tiletamine 125 mg/ml and Zolazepam 125 mg/ml, Virbac, France). Additionally, Mydrien-P eye drops® (phenylephrine hydro-chloride 5 mg/ml and tropicamide 5 mg/ml, Santen, Japan) were applied to dilate the pupils. Additionally, Alcaine® (proparacaine hydrochloride 0.5%, Alcon Laboratories Inc.) was instilled for eye anesthesia. The anesthetized rat was stably fixed on an experimental animal support stand and maintained in a position for highly reproducible electrophysiological testing.
망막전위도 검사는 Ganzfeld ERG system(Phoenix Research Labs)을 이용하여 빛 자극 및 전기 신호 측정을 시행하였다. 망막전위도 신호는 실험동물 쥐 양안을 활용하였으며, 빛의 강도를 높여가면서 순차적으로 전기 신호를 측정하였다. 빛 노출 시간은 10 msec으로 하였고, 각 빛의 강도에서 10 회의 평균값을 이용해서 전기 신호를 기록하였다. 순금으로 제작된 각막 전극을 각막 주변에 위치하였고, 기준 전극과 접지 전극을 두피와 꼬리 피하에 위치시켰다. 망막의 간세포(rod cell)의 기능을 확인하기 위하여 암순응 a파와 전반적인 망막 기능 확인을 위해 b파를 확인하였다. 도 6 및 도 7에 이종교배된 랫의 망막 전위도 검사 결과를 나타낸다. 도 6은 이종교배된 랫이 6주령일 때의 검사 결과이고, 도 7은 이종교배된 랫이 10주령일 때의 검사 결과이다. 도 6 및 도 7에서 KO ROD는 이종교배된 랫의 망막 간세포이고, WT ROD는 야생형 LE 랫의 망막 간세포이다.Electroretinography was performed by measuring light stimulation and electrical signals using the Ganzfeld ERG system (Phoenix Research Labs). Both eyes of the experimental animal rat were used for electroretinogram signals, and electrical signals were measured sequentially while increasing the intensity of light. The light exposure time was 10 msec, and the electrical signal was recorded using the average value of 10 times at each light intensity. Corneal electrodes made of pure gold were placed around the cornea, and reference electrodes and ground electrodes were placed under the skin of the scalp and tail. The dark adaptation a-wave was checked to check the function of retinal rod cells, and the b-wave was checked to check overall retinal function. Figures 6 and 7 show the electroretinogram test results of crossbred rats. Figure 6 shows the test results when a cross-bred rat was 6 weeks old, and Figure 7 shows the test results when a cross-bred rat was 10 weeks old. In Figures 6 and 7, KO ROD is a retinal stem cell of a crossbred rat, and WT ROD is a retinal stem cell of a wild-type LE rat.
그 결과, 망막전위를 통한 망막 기능 검사에서 6주령 이종교배된 랫이 야생형 LE보다 전위도 진폭이 감소하는 소견을 보였고, 시간이 경과할수록 전위도 진폭이 현저히 감소하여 망막 기능이 손상된 것으로 확인되었다(도 6 및 도 7 참조).As a result, in a retinal function test using electroretinography, 6-week-old crossbred rats showed a decrease in electromagnetism amplitude compared to wild-type LE, and it was confirmed that retinal function was damaged as electromagnetism amplitude significantly decreased over time ( 6 and 7).
실시예 3. 조직 병리소견 확인Example 3. Confirmation of tissue pathology findings
3-1. 인간, PDE6B KO 랫 및 야생형 LE 랫의 안구 조직 비교3-1. Comparison of eye tissue in humans, PDE6B KO rats and wild-type LE rats.
인간의 안구 조직과 동물 모델로 사용되어 온 PDE6B KO SD 랫 및 야생형 LE 랫의 안구 조직을 비교하였다. 인간의 안구 조직에 대한 조직 검사는 별도로 수행하지 않고, 기존에 수행되었던 연구에서 얻어진 안과질환이 없는 사람의 조직 검사 결과를 대신 사용하였다. 랫은 각 개체로부터 안구를 얻고 안구 조직을 고정하여 표본을 제작하고 조직학적인 관찰을 수행하였다.Human eye tissue was compared with the eye tissue of PDE6B KO SD rats and wild-type LE rats, which have been used as animal models. A separate biopsy of human eye tissue was not performed, and biopsy results from a person without ocular disease obtained from a previously conducted study were used instead. Rat eyes were obtained from each individual, eye tissue was fixed, specimens were prepared, and histological observations were performed.
실험용 쥐의 복강 내에 Zoletil® 0.01 ml (Tiletamine 125 ㎎/ml and Zolazepam 125 ㎎/ml, Virbac, France)을 주사하여 마취한 다음, 안구를 적출하여 4% 파라포름알데히드 용액(pH 7.4)에서 고정하였다. 고정된 안구는 시신경을 중심으로 절개하여 파라핀에 포매한 후, 4 ㎛ 두께로 잘라 H&E 염색(hematoxylin-eosin staining)을 수행하여, 조직화학적 표본을 제작하였다. 제작된 조직 표본을 정상 대조군과 비교하여 조직 병리적인 소견을 확인하였다. 도 8에 인간, PDE6B KO 랫 및 야생형 LE 랫의 안구 조직 검사 결과를 나타낸다. 도 8에서 Histology of human retina은 인간의 망막이고, PDE6B KO SD은 PDE6B KO 랫의 망막이며, LE는 야생형 LE 랫의 망막이다.Experimental rats were anesthetized by injecting 0.01 ml of Zoletil® (Tiletamine 125 mg/ml and Zolazepam 125 mg/ml, Virbac, France) into the abdominal cavity, and then the eyes were removed and fixed in 4% paraformaldehyde solution (pH 7.4). . The fixed eye was cut around the optic nerve and embedded in paraffin, then cut into 4 ㎛ thick pieces and subjected to H&E staining (hematoxylin-eosin staining) to prepare histochemical specimens. The prepared tissue samples were compared with normal controls to confirm histopathological findings. Figure 8 shows the results of eye tissue examination of humans, PDE6B KO rats, and wild-type LE rats. In Figure 8, Histology of human retina is the human retina, PDE6B KO SD is the retina of a PDE6B KO rat, and LE is the retina of a wild-type LE rat.
그 결과, 인간과 야생형 LE 랫은 도 8에서 붉은색으로 강조 표시한 것과 같이 망막색소상피층이 존재하지만, PDE6B KO 랫은 망막색소상피층이 소실된 것으로 확인되었다. 이와 같이 PDE6B KO SD 랫은 안구내에 색소가 없어서 안과 질환 연구시에 수행되는 이미징 검사 및 망막기능 검사에는 적합하지 않다는 것을 알 수 있었다(도 8 참조).As a result, it was confirmed that humans and wild-type LE rats had a retinal pigment epithelial layer, as highlighted in red in Figure 8, but that the PDE6B KO rat had lost the retinal pigment epithelial layer. In this way, it was found that PDE6B KO SD rats do not have pigment in the eye, so they are not suitable for imaging tests and retinal function tests performed when studying ocular diseases (see Figure 8).
3-2. 이종교배된 랫과 야생형 LE 랫 안구 조직 비교3-2. Comparison of eye tissue between outbred rats and wild-type LE rats.
본 발명에서 제작한 이종교배된 랫을 망막변성의 동물 모델로서 사용할 수 있는지 확인하기 위해, 안구 조직을 고정하여 표본을 제작하고 조직화학적인 관찰을 수행하였다.In order to confirm whether the crossbred rat produced in the present invention can be used as an animal model of retinal degeneration, eye tissue was fixed, specimens were prepared, and histochemical observations were performed.
상기 실시예 1-1에서 제조된 이종교배된 랫이 5주령일 때, 조직 병리 소견을 확인하였다. 정상 대조군은 야생형 LE 랫으로 하였다. 실험용 쥐의 복강 내에 Zoletil® 0.01 ml (Tiletamine 125 ㎎/ml and Zolazepam 125 ㎎/ml, Virbac, France)을 주사하여 마취한 다음, 안구를 적출하여 4% 파라포름알데히드 용액(pH 7.4)에서 고정하였다. 고정된 안구는 시신경을 중심으로 절개하여 파라핀에 포매한 후, 4 ㎛ 두께로 잘라 H&E 염색(hematoxylin-eosin staining)을 수행하여, 조직 표본을 제작하였다. 제작된 조직 표본을 정상 대조군과 비교하여 조직 병리적인 소견을 확인하였다. 도 9에 이종교배된 랫(KO)과 야생형 LE 랫(WT)이 5주령일 때의 조직화학 검사 결과를 나타낸다. KO는 이종교배된 랫이고, WT는 야생형 LE 랫이다.When the crossbred rat prepared in Example 1-1 was 5 weeks old, histopathological findings were confirmed. The normal control group was wild-type LE rats. Experimental rats were anesthetized by injecting 0.01 ml of Zoletil® (Tiletamine 125 mg/ml and Zolazepam 125 mg/ml, Virbac, France) into the abdominal cavity, and then the eyes were removed and fixed in 4% paraformaldehyde solution (pH 7.4). . The fixed eye was cut around the optic nerve and embedded in paraffin, then cut into 4 ㎛ thick pieces and subjected to H&E staining (hematoxylin-eosin staining) to prepare tissue specimens. The prepared tissue samples were compared with normal controls to confirm histopathological findings. Figure 9 shows the results of histochemical examination of crossbred rats (KO) and wild-type LE rats (WT) at 5 weeks of age. KO is an outbred rat, and WT is a wild-type LE rat.
그 결과, 이종교배된 랫에서 시간의 경과에 따라 망막의 조직 변화가 나타나는 것을 확인하였다. 이종교배된 랫은 야생형 LE 랫과 비교하여 전반적인 망막 두께가 감소되고, 외핵층(오른쪽 이미지에 Ι로 표시된 부분)이 소실되었으며, 특히 간세포, 추세포와 같은 망막하 시세포 손상의 정도가 심한 양상을 보였다. As a result, it was confirmed that retinal tissue changes appeared over time in crossbred rats. Compared to wild-type LE rats, crossbred rats showed decreased overall retinal thickness, loss of the outer nuclear layer (part marked Ι in the image on the right), and especially severe damage to subretinal photoreceptor cells such as hepatocytes and trabeculae. .
이종교배된 랫은 기존에 제조되었던 PDE6B KO SD 랫의 망막 변성과 유사한 소견을 보였고, 기존의 PDE6B KO SD 랫에서는 뚜렷하게 관찰되지 않는 망막색소상피층(retinal pigmented epithelium) 및 맥락막 색소층(choroid)이 뚜렷하게 확인되었다(도 9 참조). 이와 같이 이종교배된 랫은 망막 변성 양상을 보이면서 망막색소상피층과 맥락막 색소층이 뚜렷하게 확인되므로 망막색소변성증에 대한 동물 모델로서 사용할 수 있는 것을 확인하였다.The crossbred rats showed similar findings to the retinal degeneration of the previously prepared PDE6B KO SD rats, and the retinal pigmented epithelium and choroid, which were not clearly observed in the existing PDE6B KO SD rats, were clearly visible. This was confirmed (see Figure 9). In this way, it was confirmed that the cross-bred rat showed retinal degeneration and could be used as an animal model for retinitis pigmentosa because the retinal pigment epithelial layer and choroidal pigment layer were clearly identified.
<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Method for producing animal model with retinal degeneration <130> FPD/202104-0036/C <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F2 <400> 1 atgggaaccc cacctttgcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R5 <400> 2 gacgctctct tgcatgtcct 20 <110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Method for producing animal model with retinal degeneration <130> FPD/202104-0036/C <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F2 <400> 1 atgggaaccc cacctttgcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>R5 <400> 2 gacgctctct tgcatgtcct 20
Claims (16)
b) F1 중에서 이형접합체를 육안으로 선별하고, 상기 선별은 털 색이 야생형 LE 랫의 털 색인 개체를 선별하는 것인 단계;
c) 선별된 F1 이형접합체와 야생형 LE 랫을 역교배하여 자손 B1을 얻는 단계;
d) B1 중에서 털 색이 야생형 LE 랫의 털 색을 나타내고, 안구의 색이 흑색인 개체를 육안으로 1차 선별하는 단계;
e) 1차 선별된 B1 중에서 털 색(+/+)인 개체를 2차 선별하고, 2차 선별된 개체 중에서 망막 변성에 관여하는 유전자(+/-)인 개체를 3차 선별하는 단계;
f) 3차 선별된 B1 개체를 야생형 LE 랫과 교배하여 자손 B2를 얻는 단계를 포함하는, 망막변성증 동물 모델의 제조 방법.a) Obtaining offspring F1 by crossing an SD rat in which a gene involved in retinal degeneration has been knocked out and a wild-type LE rat;
b) visually selecting heterozygotes among F1, and selecting individuals whose fur color is that of a wild-type LE rat;
c) backcrossing the selected F1 heterozygote and wild-type LE rats to obtain progeny B1;
d) primary visual selection of individuals among B1 whose fur color represents that of a wild-type LE rat and whose eye color is black;
e) secondly selecting individuals with fur color (+/+) among the firstly selected B1, and thirdly selecting individuals with genes involved in retinal degeneration (+/-) among the secondly selected individuals;
f) A method for producing a retinal degeneration animal model, comprising the step of crossing the third selected B1 individual with a wild-type LE rat to obtain progeny B2.
망막변성증이 망막색소변성증인, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
A method for producing a retinal degeneration animal model, wherein the retinopathy is retinitis pigmentosa.
망막 변성은 망막색소변성, 혈관무늬병증, 드루젠 및 황반 변성으로 이루어진 군으로부터 선택되는 어느 하나 이상인, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
A method of producing an animal model of retinal degeneration, wherein the retinal degeneration is at least one selected from the group consisting of retinitis pigmentosa, angiopathy, drusen, and macular degeneration.
e) 단계에서 1차 선별된 B1 중에서 털 색(+/+)인 개체의 선별은 1차 선별된 B1 랫(♀)과 SD(알비노) 랫(♂)을 교배하여 야생형 LE 랫과 같은 털 색을 가지는 자손만을 생산하는 B1 개체 모체를 선별하는 것인 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
In step e), individuals with fur color (+/+) among the first selected B1 are selected by crossing the first selected B1 rat (♀) with SD (albino) rat (♂) to obtain the same fur color as the wild-type LE rat. A method for producing a retinal degeneration animal model, which involves selecting B1 individual mothers that produce only offspring having .
e) 단계에서 망막 변성에 관여하는 유전자(+/-)인 개체의 선별은 망막 변성에 관여하는 유전자 검출용 프라이머 세트를 이용하는 것인, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
A method for producing a retinal degeneration animal model, wherein in step e), the selection of individuals with genes (+/-) involved in retinal degeneration uses a primer set for detecting genes involved in retinal degeneration.
망막 변성에 관여하는 유전자는 PDE6B인, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
A method for producing an animal model of retinal degeneration, wherein the gene involved in retinal degeneration is PDE6B.
상기 PDE6B 유전자 검출용 프라이머 세트는 서열번호 1의 염기 서열로 이루어지는 정방향 프라이머; 및 서열번호 2의 염기 서열로 이루어지는 역방향 프라이머를 포함하는, 망막변성증 동물 모델의 제조 방법.In clause 7,
The primer set for detecting the PDE6B gene includes a forward primer consisting of the base sequence of SEQ ID NO: 1; And a method for producing a retinal degeneration animal model, comprising a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.
c) 단계 내지 f) 단계는 반복되며, B3 이상의 자손까지 진행되는 것인, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
Steps c) to f) are repeated and progress to B3 or higher offspring.
망막변성증 동물 모델의 제조 방법에 의해 얻어진 망막변성증 동물 모델의 안구 조직 내에는 망막색소상피층 및 맥락막 색소층 중 적어도 하나가 존재하는, 망막변성증 동물 모델의 제조 방법.According to paragraph 1,
A method for producing a retinal degeneration animal model, wherein at least one of a retinal pigment epithelial layer and a choroidal pigment layer is present in the ocular tissue of the retinal degeneration animal model obtained by the method for producing a retinal degeneration animal model.
안구 조직 내에 망막색소상피층 및 맥락막 색소층 중 적어도 하나가 존재하는, 망막변성증 동물 모델.According to clause 11,
An animal model of retinal degeneration, wherein at least one of a retinal pigment epithelial layer and a choroidal pigment layer exists in eye tissue.
망막변성증이 망막색소변성증인, 망막변성증 동물 모델.According to clause 11,
An animal model of retinal degeneration, in which retinopathy is retinitis pigmentosa.
ii) 단계 i)에서 피검 물질이 처리된 망막변성증 동물 모델을 무처리 대조군과 비교하여 망막변성증 증상의 개선 여부를 확인하는 단계를 포함하는, 망막변성증의 치료제 스크리닝 방법.i) treating the test material to the retinal degeneration animal model of claim 11; and
ii) A screening method for a treatment for retinal degeneration, comprising the step of comparing the retinal degeneration animal model treated with the test substance in step i) with an untreated control group to determine whether symptoms of retinal degeneration are improved.
상기 단계 ii)의 망막변성증 증상의 개선은, 망막 혈관 형태의 개선, 망막 단층 두께의 증가, 망막 전위도의 증가 및 망막 조직 내 세포수 증가로 이루어진 군으로부터 선택되는 어느 하나 이상인, 망막변성증의 치료제 스크리닝 방법.According to clause 14,
The improvement of the symptoms of retinal degeneration in step ii) is one or more selected from the group consisting of improvement of retinal vascular morphology, increase in retinal monolayer thickness, increase in electroretinogram, and increase in the number of cells in retinal tissue, a treatment for retinal degeneration. Screening method.
상기 피검 물질은 화합물, 추출물, 유전자 치료제 및 세포 치료제로 이루어진 군으로부터 선택되는 어느 하나 이상인, 망막변성증의 치료제 스크리닝 방법.
According to clause 14,
A method for screening a treatment for retinal degeneration, wherein the test substance is at least one selected from the group consisting of a compound, extract, gene therapy, and cell therapy.
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