KR102351161B1 - RNF220 knock-out transgenic animal model and using thereof - Google Patents
RNF220 knock-out transgenic animal model and using thereof Download PDFInfo
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Abstract
본 발명은 RNF220 유전자를 녹아웃(knock-out)시킨 형질전환 동물모델 및 이의 용도에 관한 것으로, 발달장애, 지적장애, 운동장애, 근위축측삭경화증 (ALS) 후보유전자인 RNF220 유전자 녹아웃(knock-out) 제브라피쉬(zebrafish)를 제작한 결과, 상기 RNF220 유전자 녹아웃 제브라피쉬가 운동장애 및 안구운동 장애 증상을 갖고, V2a/b 인터뉴런, Glutamate성 뉴런의 발현이 감소되는 것을 관찰하였으며, RNF220 유전자 녹아웃 제브라피쉬에 발달장애, 지적장애, 운동장애, 근위축측삭경화증 (ALS) 환자에서 발견된 다양한 점돌연변이 RNF220 유전자를 도입한 복원실험을 통하여, 유전자 기능분석 및 생물학적인 검증이 가능함을 확인함으로써, 본 발명의 RNF220 유전자 녹아웃 제브라피쉬는 발달장애, 지적장애, 운동장애, 근위축측삭경화증 (ALS) 질환 관련 약물 스크리닝 방법에 유용하게 사용될 수 있다. The present invention relates to a transgenic animal model in which the RNF220 gene is knocked out and its use, and the RNF220 gene knockout (knock-out) gene for developmental disability, intellectual disability, movement disorder, and amyotrophic lateral sclerosis (ALS) candidate gene ) As a result of producing zebrafish, it was observed that the RNF220 gene knockout zebrafish had movement disorders and eye movement disorders, and the expression of V2a/b interneurons and glutamate neurons was reduced, and RNF220 gene knockout zebrafish The present invention by confirming that gene function analysis and biological verification are possible through restoration experiments in which various point mutation RNF220 genes found in patients with developmental disabilities, intellectual disabilities, movement disorders, and amyotrophic lateral sclerosis (ALS) were introduced into fish The RNF220 gene knockout zebrafish can be usefully used for drug screening methods related to developmental disabilities, intellectual disabilities, movement disorders, and amyotrophic lateral sclerosis (ALS) diseases.
Description
본 발명은 RNF220 유전자를 녹아웃(knock-out)시킨 형질전환 동물모델 및 이의 용도에 관한 것이다.The present invention relates to a transgenic animal model in which the RNF220 gene is knocked out and uses thereof.
근위축측삭경화증 (amyotrophic lateral sclerosis, ALS)와 척수근위축증 (Spinal muscular atrophy, SMA)는 하위 운동신경세포 (motor neuron)의 퇴행을 특징으로 하는 중증 신경계 질환이다. 근위축측삭경화증과 척수근위축증 환자는 운동신경퇴행과 함께 근육 손실을 겪고, 결국 사망에 이르게 된다.Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA) are severe neurological diseases characterized by degeneration of lower motor neurons. Patients with amyotrophic lateral sclerosis and spinal muscular atrophy suffer muscle loss along with motor neurodegeneration, which ultimately leads to death.
근위축측삭경화증의 발병률은 100,000명당 2.2 ~ 2.8명으로 보고되어 있고, 환자의 약 10%가 가족성이고 나머지 90%는 산발성이라는 것이 알려져 있고, 1993년에 SOD1 (superoxide dismutase 1)이 근위축성 측삭경화증과 연관되어 있는 첫번째 유전자로 밝혀진 이래로 최근의 유전체 분석 기술의 발전과 함께 현재는 30개 이상의 유전자가 근위축측삭경화증과 연관되어 있음이 보고되고 있다 (Mathis S, 2019). 한편, 척수근위축증은 단일유전자성 질환으로 신생아 10,000명당 1명으로 발병하며, 현재까지 보고된 환자의 95%에서 SMN1 유전자의 결실 혹은 돌연변이가 그 원인으로 알려져 있다 (Lefebvre, 1995).The incidence of amyotrophic lateral sclerosis is reported to be 2.2 to 2.8 per 100,000, and it is known that about 10% of patients are familial and the remaining 90% are sporadic. Since it was identified as the first gene associated with sclerosis, with recent advances in genome analysis technology, more than 30 genes have been reported to be associated with amyotrophic lateral sclerosis (Mathis S, 2019). On the other hand, spinal muscular atrophy is a monogenetic disease, with an incidence of 1 in 10,000 newborns, and deletion or mutation of the SMN1 gene is known to be the cause in 95% of the reported cases (Lefebvre, 1995).
근위축측삭경화증 원인유전자를 밝히기 위한 세계적인 협력체제인 ALSGEN 컨소시엄은 근위축측삭경화증의 발병시기와 질병취약성에 영향을 주는 유전변이를 찾기 위해 근위축측삭경화증 환자 4252명과 대조군 5123명에 대한 유전체 메타분석을 진행하였다. 이를 통해 여섯개의 유전체 영역이 발병시기와 연관되어 있음을 밝혔고, 이 중에서, 1p34.1 유전체 영역이 가장 강하게 연관된 영역임을 확인하였다 (Ahmeti KB, 2013).The ALSGEN Consortium, a global cooperative system to uncover the causative genes of amyotrophic lateral sclerosis, conducted a genome meta-analysis of 4252 amyotrophic lateral sclerosis patients and 5123 controls to find genetic mutations that affect the onset time and disease susceptibility of amyotrophic lateral sclerosis. proceeded. Through this, it was revealed that six genomic regions were associated with the onset period, and among them, the 1p34.1 genomic region was confirmed to be the most strongly related region (Ahmeti KB, 2013).
근위축측삭경화증과 척수근위축증은 주로 운동뉴런 자체적 결함에 의해 발병하는 것으로 설명되어 왔으나, 최근에는 인터뉴런의 기능결함에 의한 운동뉴런의 기능장애과 퇴행도 ALS의 잠재적인 원인으로서 보고되고 있다 (Simone C, 2016, Clark RM, 2018).Amyotrophic lateral sclerosis and spinal muscular atrophy have been mainly described as being caused by motor neuron own defects, but recently, motor neuron dysfunction and degeneration due to interneuron dysfunction have also been reported as potential causes of ALS (Simone C. , 2016, Clark RM, 2018).
RNF220은 X-성염색체 연관 지적장애(X-linked intellectual disability, XLID)의 하나인 Miles-Carpenter Syndrome(MCS)의 원인유전자로 밝혀진 ZC4H2와 결합하는 단백질로 보고되어 있다. ZC4H2 유전자가 결손되었을 때, Miles-Carpenter Syndrome 환자의 증상과 매우 유사한 운동장애 증상이 나타났다. 현재까지 운동장애 증상에 대한 주요 원인으로 근육 발달의 이상이나 운동 신경세포 (motor neuron)의 소멸에 의한 뇌 혹은 척수의 기능 이상이 알려져 있다. 그러나, 운동장애를 보이는 ZC4H2 유전자 녹아웃 동물에서 운동 신경세포는 정상적으로 발생하였으나, V2a/b 인터뉴런은 그 발현이 크게 감소되어 있다는 것이 보고되어 있다. 이러한 결과는 운동장애의 원인으로 근육 발달의 이상이나 운동 신경세포 이상 외에, 운동 신경세포의 작동을 조절하는 인터뉴런도 관여하고 있음을 보여주고 있다(대한민국 공개특허 제10-2016-0143913호). RNF220은 발생 중인 신경관 (developing neural tube)에서 발현하고 있으나, RNF220 유전자를 결손시켰을 때 근위축측삭경화증 및 척수근위축증과 같은 운동장애 증상이 나타나는지에 대해서는 알려져 있지 않다. 본 발명에서는 발달장애, 지적장애, 운동장애의 원인유전자인 ZC4H2와 결합하는 새로운 인자인 RNF220가 근위축측삭경화증 (ALS)의 원인유전자가 존재할 것으로 예상되는 염색체 1p34.1에 위치한다는 점에 착안하여, RNF220가 운동장애 및 근위축측삭경화증 (ALS)의 새로운 원인유전자임을 RNF220 유전자 녹아웃 제브라피쉬 기능분석을 통하여 규명하였다.RNF220 has been reported as a protein binding to ZC4H2, which has been identified as the causative gene of Miles-Carpenter Syndrome (MCS), one of X-linked intellectual disability (XLID). When the ZC4H2 gene was deleted, symptoms of movement disorders very similar to those of Miles-Carpenter Syndrome patients appeared. Until now, as the main cause of movement disorder symptoms, abnormal muscle development or brain or spinal cord function abnormalities due to the disappearance of motor neurons are known. However, it has been reported that motor neurons developed normally in ZC4H2 gene knockout animals showing movement disorders, but the expression of V2a/b interneurons was greatly reduced. These results show that, as a cause of movement disorders, interneurons that control the operation of motor neurons are involved in addition to abnormalities in muscle development or abnormalities in motor neurons (Korean Patent Application Laid-Open No. 10-2016-0143913). Although RNF220 is expressed in a developing neural tube, it is not known whether movement disorders such as amyotrophic lateral sclerosis and spinal muscular atrophy occur when the RNF220 gene is deleted. In the present invention, RNF220, a new factor that binds to ZC4H2, a causative gene for developmental disability, intellectual disability, and movement disorder, is located on chromosome 1p34.1 where the causative gene for amyotrophic lateral sclerosis (ALS) is expected to exist. , It was identified through functional analysis of RNF220 gene knockout zebrafish that RNF220 is a novel causative gene of movement disorders and amyotrophic lateral sclerosis (ALS).
제브라피쉬 (zebrafish)는 체외 수정을 하고, 발생배가 투명하고, 대량의 수정란을 쉽게 확보할 수 있고, 초기 발생이 매우 빨라 대부분의 조직 및 장기가 하루 만에 형성된다. 제브라피쉬 전체 유전체 정보는 현재 모두 공개되어 있어 genome 데이터베이스를 통해 쉽게 검색할 수 있고, 척추동물로서 유전체 구성이 인간과 매우 비슷하다. 상기의 특징으로 형질전환 제브라피쉬에 관한 연구는 꾸준히 진행되어 왔고, 특히 최근에는 CRISPR-Cas9 유전자 가위를 활용한 유전자 녹아웃 제브라피쉬의 제작이 매우 활발히 진행되고 있다. Zebrafish perform in vitro fertilization, have a transparent embryonic embryo, and can easily obtain a large number of fertilized eggs. The entire genome information of the zebrafish is currently publicly available, so it can be easily searched through the genome database. As a vertebrate, its genome is very similar to that of humans. With the above characteristics, research on transgenic zebrafish has been steadily progressing, and in particular, recently, gene knockout zebrafish using CRISPR-Cas9 gene scissors is being actively produced.
인위적으로 외부의 유전자를 삽입한 형질전환 동물(transgenic animals) 또는 유전자를 제거한 녹아웃 동물 (knock-out animals)은, 생명체에서의 특정 유전자의 역할을 규명하는 기초연구에 유용하게 쓰일 수 있다. 특히, 특정 질병의 원인이 되는 유전자를 삽입 또는 결실시킨 형질전환 동물 또는 유전자 녹아웃 동물은 인간의 질병을 모델링하는 모델동물로서 사용할 수 있다. 즉, 특정 질병의 발병기전을 파악하거나, 치료제 후보물질의 약물로서의 가능성을 알아보는데 매우 유용하다. Transgenic animals in which external genes are artificially inserted or knock-out animals in which genes are removed can be usefully used in basic research to identify the role of specific genes in living things. In particular, a transgenic animal or a gene knockout animal in which a gene causing a specific disease is inserted or deleted can be used as a model animal for modeling a human disease. That is, it is very useful to understand the pathogenesis of a specific disease or to find out the potential of a therapeutic candidate substance as a drug.
이에 본 발명자들은 쉽고 빠르게 접근할 수 있는 인간의 질환 모델을 개발하기 위해 노력하던 중, 근위축측삭경화증 (ALS) 원인의 후보유전자인 RNF220 유전자 녹아웃 제브라피쉬가 운동 장애 증상을 갖고, V2a/b 인터뉴런 및 glutamate성(glutamatergic) 뉴런의 발생이 감소되는 것을 확인하였으며, RNF220 유전자 녹아웃 제브라피쉬에 RNF220 유전자를 복원하였을 때, V2a 뉴런의 발생이 회복됨을 확인함으로써 본 발명을 완성하였다.Therefore, while the present inventors were trying to develop a human disease model that can be easily and quickly accessed, the RNF220 gene knockout zebrafish, a candidate gene for amyotrophic lateral sclerosis (ALS), has movement disorder symptoms, and V2a/b inter It was confirmed that the generation of neurons and glutamatergic neurons was reduced, and when the RNF220 gene was restored in RNF220 gene knockout zebrafish, the present invention was completed by confirming that the generation of V2a neurons was restored.
본 발명의 목적은 RNF220 유전자를 녹아웃(knock-out)시킨 형질전환 동물모델 및 이의 용도를 제공하는 것이다.It is an object of the present invention to provide a transgenic animal model in which the RNF220 gene is knocked out and uses thereof.
상기 목적을 달성하기 위하여, 본 발명은 RNF220 유전자가 녹아웃(knock-out)된 형질전환 동물을 제공한다.In order to achieve the above object, the present invention provides a transgenic animal in which the RNF220 gene is knocked out.
또한, 본 발명은Also, the present invention
1) RNF220 유전자 녹아웃(knock-out) 콘스트럭트를 제조하는 단계;1) preparing an RNF220 gene knock-out construct;
2) 상기 단계 1)의 콘스트럭트를 수정란에 도입하는 단계를 포함하는 형질전환 동물의 제조방법을 제공한다.2) It provides a method for producing a transgenic animal comprising the step of introducing the construct of step 1) into a fertilized egg.
또한, 본 발명은 RNF220 유전자가 녹아웃된 형질전환 수정란을 제공한다.In addition, the present invention provides a transgenic fertilized egg in which the RNF220 gene is knocked out.
또한, 본 발명은,In addition, the present invention,
1) 본 발명의 형질전환 동물에 피검화합물을 처리하는 단계 및1) treating the test compound in the transgenic animal of the present invention; and
2) 상기 단계 1)의 피검화합물이 처리된 형질전환 동물을 무처리된 대조군과 비교하여 증상 회복여부를 판단하는 단계를 포함하는 운동장애, 근위축측삭경화증(ALS) 관련 질환 약물 스크리닝 방법을 제공한다.2) A drug screening method for movement disorders and amyotrophic lateral sclerosis (ALS)-related diseases comprising the step of comparing the transgenic animals treated with the test compound of step 1) with the untreated control group to determine whether symptoms have recovered do.
본 발명은 RNF220 유전자 녹아웃(knock-out) 제브라피쉬 (zebrafish)를 제작한 결과, 상기 RNF220 유전자 녹아웃 제브라피쉬가 운동장애 증상을 갖고, V2a/b 인터뉴런, Glutamate 성 뉴런의 발현이 감소되는 것을 관찰하였으며, RNF220 유전자 녹아웃 제브라피쉬에 RNF220 유전자를 복원하였을 때, V2a 인터뉴런의 발현이 회복됨을 확인함으로써, 본 발명의 RNF220 유전자 녹아웃 제브라피쉬는 운동장애, 근위축측삭경화증 (ALS) 질환 관련 동물모델로 유용하게 사용될 수 있다.As a result of producing RNF220 gene knock-out zebrafish, the present invention observed that the RNF220 gene knock-out zebrafish had ataxia symptoms, and the expression of V2a/b interneurons and glutamate neurons was reduced. When the RNF220 gene was restored in the RNF220 gene knockout zebrafish, it was confirmed that the expression of the V2a interneuron was restored. It can be useful.
도 1은 인간, 마우스 및 제브라피쉬의 RNF220 아미노산 서열 비교를 나타낸 도이다.
도 2A 및 2B는 24 hpf와 72 hpf 제브라피쉬에서 RNF220의 시간적 및 공간적 발현양상을 나타낸 도이다.
도 3은 CRISPR-Cas9 유전자가위를 이용한 RNF220 녹아웃 콘스트럭트 제작 설계 및 RNF220 유전자 녹아웃 제브라피쉬의 외형적 표현형을 나타낸 도이다.
도 3A와 3B는 엑손 12의 CRISPR-Cas9 유전자가위의 돌연변이 타겟 사이트와 유전자 가위에 의해 유발된 돌연변이를 나타낸 도이다.
도 3C는 RNF220 유전자 녹아웃 제브라피쉬를 판별하는 PCR 결과를 전기영동 결과를 통해 나타낸 도이다.
도 3D와 3E는 80 hpf에서 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬 외형을 비교한 도이다.
도 3F와 3G는 8 dpf에서 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬의 외형을 비교한 도이다.
도 4는 RNF220 유전자 녹아웃 제브라피쉬의 운동장애 증상을 나타낸 도이다.
도 4A 및 4C는 RNF220 유전자 녹아웃 제브라피쉬의 비정상적인 안구운동을 나타낸 도이다.
도 4B 및 4D는 RNF220 유전자 녹아웃 제브라피쉬의 비정상적인 자유수영을 나타낸 도이다.
도 5는 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬의 운동뉴런, 세포죽음, 근육 발생을 나타낸 도이다.
도 5A 및 5B는 운동뉴런을 표지하는 isl1 유전자의 발현을 나타낸 도이다.
도 5C 및 5D는 운동뉴런과 정상적인 축삭돌기를 표지하는 Znp1 단백질의 발현을 나타낸 도이다.
도 5E 및 5F는 세포죽음을 표지하는 caspase 3 단백질의 발현을 나타낸 도이다.
도 5G 및 5H는 근육세포를 표지하는 Myosin-1 (A4.1025) 단백질의 발현을 나타낸 도이다.
도 6은 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬의 인터뉴런 발현 양상을 나타낸 도이다.
도 6A 및 6B는 V0 인터뉴런을 표지하는 evx1의 발현을 나타낸 도이다.
도 6C 및 6D는 V1 인터뉴런을 표지하는 en1b의 발현을 나타낸 도이다.
도 6E, 6F, 6G, 6H는 V2a/b 인터뉴런 전구체 및 V2a 인터뉴런을 표지하는 vsx1 및 vsx2의 발현을 나타낸 도이다.
도 7은 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬의 뇌에서 V2a인터뉴런, GABA성 뉴런, Glutamate성 뉴런의 발현을 나타낸 도이다.
도 7A 및 7B는 후뇌에서 V2a 인터뉴런을 표지하는 vsx2의 발현을 나타낸 도이다.
도 7C 및 7D는 Glutamate성 뉴런을 표지하는 slc17a6b의 발현을 나타낸 도이다.
도 7E 및 7F는 GABA성 뉴런을 표지하는 gad1b의 발현을 나타낸 도이다.
도 8은 정상과 RNF220 유전자 녹아웃 제브라피쉬에 RNF220 유전자를 복원하였을 때, 뉴런 발현 양상을 나타낸 도이다.
도 8A 및 8B는 정상 제브라피쉬와 RNF220 유전자 녹아웃 제브라피쉬의 vsx2 발현을 나타낸 도이다.
도 8C, 8D, 8E, 8F, 8G, 8H 및 8I는 RNF220 유전자 녹아웃 제브라피쉬에 변이형 RNF220 유전자를 복원하였을 때, vsx2 발현을 나타낸 도이다.
도 8J는 RNF220 정상 제브라피쉬와 유전자 녹아웃 제브라피쉬에 RNF220 유전자를 복원하였을 때의 vsx2 발현을 정량 분석한 결과를 나타낸 도이다. 1 is a diagram showing a comparison of RNF220 amino acid sequences of human, mouse and zebrafish.
2A and 2B are diagrams showing temporal and spatial expression patterns of RNF220 in 24 hpf and 72 hpf zebrafish.
3 is a diagram illustrating the design of the RNF220 knockout construct using CRISPR-Cas9 gene scissors and the external phenotype of the RNF220 gene knockout zebrafish.
3A and 3B are diagrams showing the mutation target site of the CRISPR-Cas9 gene scissors of exon 12 and mutations induced by the gene scissors.
Figure 3C is a diagram showing the PCR results for discriminating the RNF220 gene knockout zebrafish through the electrophoresis results.
3D and 3E are diagrams comparing the appearance of normal zebrafish and RNF220 gene knockout zebrafish at 80 hpf.
3F and 3G are diagrams comparing the appearance of normal zebrafish and RNF220 gene knockout zebrafish at 8 dpf.
4 is a diagram showing the symptoms of movement disorders of RNF220 gene knockout zebrafish.
4A and 4C are diagrams showing abnormal eye movements of RNF220 gene knockout zebrafish.
4B and 4D are diagrams showing abnormal free swimming of RNF220 gene knockout zebrafish.
5 is a diagram showing motor neurons, cell death, and muscle development in normal zebrafish and RNF220 gene knockout zebrafish.
5A and 5B are diagrams showing the expression of the isl1 gene that labels motor neurons.
5C and 5D are diagrams showing the expression of Znp1 protein that labels motor neurons and normal axons.
5E and 5F are diagrams showing the expression of
5G and 5H are diagrams showing the expression of Myosin-1 (A4.1025) protein that labels muscle cells.
6 is a diagram showing interneuron expression patterns of normal zebrafish and RNF220 gene knockout zebrafish.
6A and 6B are diagrams showing the expression of evx1 that labels V0 interneurons.
6C and 6D are diagrams showing the expression of en1b that labels the V1 interneuron.
6E, 6F, 6G, and 6H are diagrams showing the expression of vsx1 and vsx2 that label V2a/b interneuron precursors and V2a interneurons.
7 is a diagram showing the expression of V2a interneurons, GABA neurons, and glutamate neurons in the brains of normal zebrafish and RNF220 gene knockout zebrafish.
7A and 7B are diagrams showing the expression of vsx2, which labels the V2a interneuron in the hindbrain.
7C and 7D are diagrams showing the expression of slc17a6b that labels glutamate neurons.
7E and 7F are diagrams showing the expression of gad1b that labels GABAergic neurons.
8 is a diagram showing a neuron expression pattern when the RNF220 gene is restored in normal and RNF220 gene knockout zebrafish.
8A and 8B are diagrams showing vsx2 expression in normal zebrafish and RNF220 gene knockout zebrafish.
8C, 8D, 8E, 8F, 8G, 8H and 8I are diagrams showing vsx2 expression when the mutant RNF220 gene is restored in RNF220 gene knockout zebrafish.
8J is a diagram showing the results of quantitative analysis of vsx2 expression when the RNF220 gene is restored in RNF220 normal zebrafish and gene knockout zebrafish.
본 발명은 RNF220 유전자가 녹아웃(knock-out)된 형질전환 동물을 제공한다.The present invention provides a transgenic animal in which the RNF220 gene is knocked out.
상기 RNF220 유전자는 표 1의 서열번호 1로 구성되는 것이 바람직하나 이에 한정하지 않는다.The RNF220 gene is preferably composed of SEQ ID NO: 1 of Table 1, but is not limited thereto.
Zebrafish RNF220 cDNA sequence(서열번호 1)
Zebrafish RNF220 cDNA sequence (SEQ ID NO: 1)
GGTGCTAGCATCGACGGCTGAGGCTAGCCGTGATGCCTCCATCCCCTGCCAACAGCCTCGGCCATTCGGTGTGCCAG
TGTCTGTGGAGAAGGACGTTCACCTGCCCTTCAACAATGGTTCCTATACGTTTGCCTCCATGTACCACCGACAGGGC
GCCGTACCACCAGGCTTCCCCAACAGGGACTTTCCTCCTTCCCTTCTACACCTCCATCATCAGTTTGCACCACCCAA
TCTGGACTGCTCCCCCATAAGCATGCTCAACCACAGTGGTGTCGGGGCCTTCCGGCCATTCGCTTCTCCTCCGGAAG
ATCGTGATGGAGCGGGTGGTGGATACCAGTCTGCATTCACCCCTGCCAAGAGGCTAAAGGGCTGCCTGGAGGCCGAG
GCGTCCCCGCACTTGCGCTACTCAGACGCAGAAGGGAAAGAATATGACTTTGGTGGTGCTCAGATCCCATCCAGCTC
ACCCAGTGCTCTCAAAGCTGTGGAAGATGCAGGGAAGAAGATCTTCGCAGTGTCAGGCCTGCTGTCAGATCGAGAAA
CCTCTTCCAGCCCAGAGGATCGCATTGAGCGCTGTAAGAAGAAAGTGTCACTCTATGACAGTCAGGCACCCATCTGC
CCCATCTGTCAGGTTCTTTTGCGTCCGGGCGAACTACAGGAGCACATGGAGCAGGAGATGGAGAGGCTCACCCACAT
GCACATCAGCAAGAACCCATCACACAAGGACATCAATGTGGCTCCAGGCACACCGAAGTCTCTCCTGTTGTCAGTGC
ACATCAAACGTGAGGGCGAGTCTCCAGTTGTGTCCCCGCTCTCCTCTGATGAAGCTCACCATTCTGACAGATACCAG
ACGTTTTTACGAGTGCGAGCTAACAGGCAAACACGATTGAATGCCCGGATTGGGAAAATGAAGCGCCGGAAGCCTGA
GGATGGACAGAGGGAAGGTGCAACAGAGGAGGATTCTGCAGATGTTGAAGGAGAGAACGGAACGCGGTTTGAAGAGT
ACGAGTGGTGCGGACAAAAGAGGGTCCGAGCTACTACATTACTGGAAGGTGGTTTCAGAGGAACAGGCTTTGCCATG
TGTAGCACGAAGGAGAGCCATGACAGTGACGCAGACCTGGACGTGGATGGAGATGACACACTGGAGTACGGCAAAGC
CCAGTACACTGAAGCAGACATCATTCCTTGCTCTGGAGAGGACCAGGGAGAGGCCAAAGAACGTGAGGCACTTCGTG
GAGCCGTTTTGAATGGTGGCGTGCCGTCCAATAGAATAACACCTGAGTTCTCCAAGTGGGCCAGTGATGAAATGCCA
TCCACAAGTAATGGCGAGAGCAGCAAACAAGAGCCCAGCTCTTCATCTTCGTCTTCCACACAGAGAACCTGTAAAAA
CAGCGAGATAGAGAAGATCACAGAGGACTCGACAGCGACCACACTGGAGGCGCTAAAGGCCCGCATAAGAGAACTGG
AGAAGCAGATCCTCAGAGGAGACCGATACAAGTGTCTCATATGCATGGACTCTTATACAATGCCACTCACCTCCATC
CAATGTTGGCACGTGCACTGTGAAGAATGCTGGCTAAGGACTCTGGGAAACAAGAAACTCTGCCCACAATGCAACAC
CATCACGTCACCAGGGGACCTCAGGCGTGTCTATTTGTGAATGGACTTGCATCGGGCGGCTTTCAAAATGGAGAGCTCCTCCTACCTACCCAACCCGTTAGCATCCCCTGCACTAAT
GGTGCTAGCATCGACGGCTGAGGCTAGCCGTGATGCCTCCATCCCCTGCCAACAGCCTCGGCCATTCGGTGTGCCAG
TGTCTGTGGAGAAGGACGTTCACCTGCCCTTCAACAATGGTTCCTATACGTTTGCCTCCATGTACCACCGACAGGGC
GCCGTACCACCAGGCTTCCCCAACAGGGACTTTCCTCCTTCCCTTCTACACCTCCATCATCAGTTTGCACCACCCAA
TCTGGACTGCTCCCCCATAAGCATGCTCAACCACAGTGGTGTCGGGGCCTTCCGGCCATTCGCTTCTCCTCCGGAAG
ATCGTGATGGAGCGGGTGGTGGATACCAGTCTGCATTCACCCCTGCCAAGAGGCTAAAGGGCTGCCTGGAGGCCGAG
GCGTCCCCGCACTTGCGCTACTCAGACGCAGAAGGGAAAGAATATGACTTTGGTGGTGCTCAGATCCCATCCAGCTC
ACCCAGTGCTCTCAAAGCTGTGGAAGATGCAGGGAAGAAGATCTTCGCAGTGTCAGGCCTGCTGTCAGATCGAGAAA
CCTCTTCCAGCCCAGAGGATCGCATTGAGCGCTGTAAGAAGAAAGTGTCACTCTATGACAGTCAGGCACCCATCTGC
CCCATCTGTCAGGTTCTTTTGCGTCCGGGCGAACTACAGGAGCACATGGAGCAGGAGATGGAGAGGCTCACCCACAT
GCACATCAGCAAGAACCCATCACACAAGGACATCAATGTGGCTCCAGGCACACCGAAGTCTCTCCTGTTGTCAGTGC
ACATCAAACGTGAGGGCGAGTCTCCAGTTGTGTCCCCGCTCTCCTCTGATGAAGCTCACCATTCTGACAGATACAG
ACGTTTTTACGAGTGCGAGCTAACAGGCAAACACGATTGAATGCCCGGATTGGAAAATGAAGCGCCGGAAGCCTGA
GGATGGACAGAGGGAAGGTGCAACAGAGGAGGATTCTGCAGATGTTGAAGGAGAGAACGGAACGCGGTTTGAAGAGT
ACGAGTGGTGCGGACAAAAGAGGGTCCGAGCTACTACATTACTGGAAGGTGGTTTCAGAGGAACAGGCTTTGCCATG
TGTAGCACGAAGGAGAGCCATGACAGTGACGCAGACCTGGACGTGGATGGAGATGACACACTGGAGTACGGCAAAGC
CCAGTACACTGAAGCAGACATCATTCCTTGCTCTGGAGAGGACCAGGGAGGCCAAAGAACGTGAGGCACTTCGTG
GAGCCGTTTTGAATGGTGGCGTGCCGTCCAATAGAATAACACCTGAGTTCTCCAAGTGGGCCAGTGATGAAATGCCA
TCCACAAGTAATGGCGAGAGCAGCAAACAAGAGCCCAGCTCTTCATCTTCGTCTTCCACACAGAGAACCTGTAAAAA
CAGCGAGATAGAGAAGATCACAGAGGACTCGACAGCGACCACACTGGAGGCGCTAAAGGCCCGCATAAGAGAACTGG
AGAAGCAGATCCTCAGAGGAGACCGATACAAGTGTCTCATATGCATGGACTCTTATACAATGCCACTCACCTCCATC
CAATGTTGGCACGTGCACTGTGAAGAATGCTGGCTAAGGACTCTGGGAAACAAGAAACTCTGCCCACAATGCAACAC
CATCACGTCACCAGGGGACCTCAGGCGTGTCTATTTGTGA
또한, 상기 형질전환 동물은 운동 장애 관련 질환을 갖는 것을 특징으로 한다.In addition, the transgenic animal is characterized in that it has a movement disorder-related disease.
상기 운동 장애 관련 질환은 연합신경세포의 발생 또는 분화의 이상으로 인한 것일 수 있다.The movement disorder-related disease may be due to abnormality in the development or differentiation of associated neurons.
상기 운동 장애 관련 질환은 발달장애, 지적장애, 운동장애 및 근위축측삭경화증(ALS)으로 이루어진 군으로부터 선택된 어느 하나인 것일 수 있다. The movement disorder-related disease may be any one selected from the group consisting of developmental disabilities, intellectual disabilities, movement disorders, and amyotrophic lateral sclerosis (ALS).
상기 형질전환 동물은 제브라피쉬인 것이 바람직하다. The transgenic animal is preferably a zebrafish.
또한, 본 발명은Also, the present invention
1) RNF220 유전자 녹아웃(knock-out) 콘스트럭트를 제조하는 단계;1) preparing an RNF220 gene knock-out construct;
2) 상기 단계 1)의 콘스트럭트를 수정란에 도입하는 단계; 를 포함하는 형질전환 동물의 제조방법을 제공한다.2) introducing the construct of step 1) into the fertilized egg; It provides a method for producing a transgenic animal comprising a.
상기 단계 1)의 녹아웃 콘스트럭트는 유전자 가위를 포함하는 것이 바람직하고, 상기 유전자 가위는 CRISPR/Cas9(clustered regulary interspaced short palindromic repeats/CRISPR-associated protein-9), ZFN(zinc-finger nuclease) 및 TALEN(transcription activator-like effector nuclease)으로 구성된 군으로부터 선택되는 어느 하나인 것이 바람직하나, 이에 한정되지 않는다.Preferably, the knockout construct of step 1) includes gene scissors, and the gene scissors include CRISPR/Cas9 (clustered regulary interspaced short palindromic repeats/CRISPR-associated protein-9), ZFN (zinc-finger nuclease), and TALEN. (transcription activator-like effector nuclease) is preferably any one selected from the group consisting of, but is not limited thereto.
또한, 상기 유전자 가위를 포함하는 콘스트럭트는 RNF220를 녹아웃시키는 것이 바람직하나, RNF220 서열 전체뿐만 아니라 서열의 일부만 녹아웃된 경우도 모두 포함한다.In addition, the construct including the gene scissors preferably knocks out RNF220, but includes all cases in which not only the entire RNF220 sequence but also a part of the sequence is knocked out.
또한, 상기 단계 2)의 수정란은 제브라피쉬(zebrafish)의 수정란인 것이 바람직하다. In addition, the fertilized egg in step 2) is preferably a fertilized egg of a zebrafish.
또한, 상기 단계 2)의 콘스트럭트를 수정란에 도입하는 방법은 유전자 컨스트럭트를 제조한 후 미세주입법(microinjection), 전기천공법(electroporation), 입자 분사법(particle bombardment)을 이용한 기법 중에서 적절하게 선택하여 형질전환 시킬 수 있다. In addition, the method of introducing the construct of step 2) into the fertilized egg is appropriate among techniques using microinjection, electroporation, and particle bombardment after preparing the gene construct. can be selected for transformation.
또한, 본 발명은 RNF220 유전자가 녹아웃된 형질전환 수정란을 제공한다.In addition, the present invention provides a transgenic fertilized egg in which the RNF220 gene is knocked out.
상기 RNF220 유전자는 서열번호 1로 구성되는 것이 바람직하나 이에 한정하지 않는다.The RNF220 gene is preferably composed of SEQ ID NO: 1, but is not limited thereto.
상기 RNF220 유전자가 녹아웃된 형질전환 수정란은 RNF220 유전자 녹아웃(knock out)콘스트럭트를 수정란에 도입하여 제조된 것일 수 있다. The transgenic fertilized egg in which the RNF220 gene is knocked out may be prepared by introducing an RNF220 gene knockout construct into the fertilized egg.
상기 녹아웃 콘스트럭트는 유전자 가위를 포함하는 것이 바람직하고, 상기 유전자 가위는 CRISPR/Cas9(clustered regulary interspaced short palindromic repeats/CRISPR-associated protein-9), ZFN(zinc-finger nuclease) 및 TALEN(transcription activator-like effector nuclease)으로 구성된 군으로부터 선택되는 어느 하나인 것이 바람직하나, 이에 한정되지 않는다.The knockout construct preferably includes gene scissors, and the gene scissors include CRISPR/Cas9 (clustered regulary interspaced short palindromic repeats/CRISPR-associated protein-9), ZFN (zinc-finger nuclease) and TALEN (transcription activator- like effector nuclease) is preferably any one selected from the group consisting of, but is not limited thereto.
또한, 상기 유전자 가위를 포함하는 콘스트럭트는 RNF220 를 녹아웃시키는 것이 바람직하나, RNF220 서열 전체뿐만 아니라 서열의 일부만 녹아웃된 경우도 모두 포함한다.In addition, the construct including the gene scissors preferably knocks out RNF220, but includes all cases in which not only the entire RNF220 sequence but also a part of the sequence is knocked out.
상기 콘스트럭트를 수정란에 도입하는 방법은 유전자 컨스트럭트를 제조한 후 미세주입법(microinjection), 전기천공법(electroporation), 입자 분사법(particle bombardment)을 이용한 기법 중에서 적절하게 선택하여 형질전환 시킬 수 있다. The method for introducing the construct into a fertilized egg is to select an appropriate method from among techniques using microinjection, electroporation, and particle bombardment after preparing the gene construct for transformation. can
또한, 본 발명은 Also, the present invention
1) 본 발명의 형질전환 동물에 피검화합물을 처리하는 단계; 및1) treating the test compound to the transgenic animal of the present invention; and
2) 상기 단계 1)의 피검화합물이 처리된 형질전환 동물을 무처리된 대조군과 비교하여 증상 회복여부를 판단하는 단계를 포함하는 운동 장애 관련 질환 약물 스크리닝 방법을 제공한다.2) It provides a drug screening method for a movement disorder-related disease comprising the step of comparing the transgenic animal treated with the test compound of step 1) with an untreated control group to determine whether symptoms are recovered.
상기 단계 1)의 피검 화합물은 천연화합물, 합성화합물, RNA, DNA, 폴리펩티드, 효소, 단백질, 리간드, 항체, 항원, 박테리아 또는 진균의 대사산물 및 생활성 분자로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 한정되지 않는다.The test compound of step 1) may be any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules. However, the present invention is not limited thereto.
상기 단계 2)의 증상 회복여부 판단은 신경세포 관련 유전자의 발현 변화 또는 이상 행동의 회복을 통해 판단한다.The determination of whether to recover the symptoms in step 2) is determined through a change in the expression of a neuron-related gene or recovery of an abnormal behavior.
상기 동물모델은 제브라피쉬인 것이 바람직하다.The animal model is preferably a zebrafish.
본 발명의 구체적인 실시예에서, 본 발명자들은 제브라피쉬로부터 RNF220 유전자를 분리하여 홀-마운트 정위치 교잡법을 수행하여 RNF220 유전자의 전사가 주로 발달 중인 신경계에서 발현하는 것을 확인하였으며(도 2 참조), CRISPR/Cas9 유전자 가위를 이용하여 RNF220 유전자가 녹아웃된 제브라피쉬를 제조하여 형태학적 표현형을 확인한 결과, RNF220 유전자가 녹아웃된 제브라피쉬와 야생형 제브라피쉬의 전반적인 발생과정에서의 차이가 없음을 확인하였다(도 3 참조). 또한, 홀-마운트 면역조직화학 염색법을 수행한 결과 RNF220 유전자 녹아웃 제브라피쉬의 운동뉴런 및 축삭돌기는 정상적인 발생을 하는 것을 확인하였다(도 5 참조). RNF220 유전자 녹아웃 제브라피쉬의 행동학적 표현형을 확인하기 위해 행동 실험을 한 결과, 자유수영의 행동에 이상이 생겼음을 확인하였고, 안구운동에도 문제가 있음을 확인하였다(도 4 참조). RNF220 유전자 녹아웃으로 인해 V2a 인터뉴런의 마커인 vsx2와 glutamate성 뉴런 마커인 slc17a6b의 발현이 크게 감소하였음을 확인하여 V2a 인터뉴런의 발생에 특이적으로 문제가 있음을 확인하였고, GABA성 인터뉴런 마커의 발현에는 차이가 없었음을 확인하였다(도 6 참조). RNF220 유전자 녹아웃 제브라피쉬에 인간의 정상 RNF220 유전자를 도입하여 복원하였을 때 vsx2의 발현이 회복됨을 확인하였다(도 8 참조). 하지만, 운동장애 환자 유래의 비정상 RNF220 유전자를 도입하였을 때는 회복이 되지 않음을 확인하였다. 따라서, 본 발명은 RNF220 녹아웃 제브라피쉬는 운동장애 관련 질환 동물모델로서의 활용이 가능하다. In a specific embodiment of the present invention, the present inventors isolated the RNF220 gene from zebrafish and performed Hall-mount in situ hybridization to confirm that the transcription of the RNF220 gene is mainly expressed in the developing nervous system (see FIG. 2), As a result of confirming the morphological phenotype by preparing zebrafish in which the RNF220 gene was knocked out using CRISPR/Cas9 gene scissors, it was confirmed that there was no difference in the overall development process between the zebrafish in which the RNF220 gene was knocked out and the wild-type zebrafish (Fig. see 3). In addition, as a result of performing Hall-mount immunohistochemical staining, it was confirmed that motor neurons and axons of the RNF220 gene knockout zebrafish had normal development (see FIG. 5 ). As a result of a behavioral experiment to confirm the behavioral phenotype of the RNF220 gene knockout zebrafish, it was confirmed that there was an abnormality in the free swimming behavior, and that there was also a problem in eye movement (see FIG. 4 ). It was confirmed that the expression of vsx2, a marker of V2a interneuron, and slc17a6b, a glutamate neuron marker, were significantly reduced due to RNF220 gene knockout, confirming that there was a specific problem in the generation of V2a interneuron, and It was confirmed that there was no difference in expression (see FIG. 6). It was confirmed that the expression of vsx2 was restored when the normal human RNF220 gene was introduced into the RNF220 gene knockout zebrafish and restored (see FIG. 8 ). However, it was confirmed that recovery was not achieved when the abnormal RNF220 gene derived from a movement disorder patient was introduced. Therefore, according to the present invention, the RNF220 knockout zebrafish can be utilized as an animal model for movement disorder-related diseases.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해서 한정되는 것은 아니다. However, the following Examples and Experimental Examples only illustrate the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
<실시예 1> 제브라피쉬 사육 및 관리<Example 1> Zebrafish breeding and management
제브라피쉬는 공지된 문헌들과 동일한 기준 조건하에서 유지 관리되었다. 야생형(wild type) 제브라피쉬를 사용하였으며, 먹이는 시판하고 있는 브라인 쉬림프(brine shrimp)를 부화시켜 사육하였다. 제브라피시는 온도 28.5℃, 오전 9시부터 오후 11시까지 점등하고, 그 외 시간 소등 조건에서 사육하였다. 상기 제브라피쉬에서 수득된 수정란은 에그 워터(egg water, Sea Salts, 60ug/ml)를 사용하여 세척하였고, 페트리 디쉬(petri dish)에서 발생시켰다. 그런 다음, 발생과정은 해부현미경을 통하여 관찰하였으며, 발생배를 시간별, 형태학적 변화에 따라 선별하여 4% 파라포름알데하이드(paraformaldehyde)/PBS로 고정하였다. 제브라피쉬와 관련된 모든 실험은 충남대학교 동물실험윤리위원회(Institutional Animal Care and Use Committees of Chungnam National University) 승인 하에 진행되었다. Zebrafish were maintained under the same standard conditions as in the known literature. Wild type zebrafish were used, and commercially available brine shrimp were hatched to feed. Zebrafish were bred at a temperature of 28.5° C., lit from 9 am to 11 pm, and turned off at other times. The fertilized eggs obtained from the zebrafish were washed using egg water (egg water, Sea Salts, 60ug/ml), and generated in a petri dish. Then, the development process was observed through a dissecting microscope, and embryonic embryos were selected according to time and morphological changes and fixed with 4% paraformaldehyde/PBS. All experiments related to zebrafish were conducted under the approval of the Institutional Animal Care and Use Committees of Chungnam National University.
<실시예 2> RNF220 유전자 발현 확인<Example 2> RNF220 gene expression confirmation
<2-1> 제브라피쉬로부터 RNF220 유전자 분리<2-1> RNF220 gene isolation from zebrafish
제브라피쉬 RNF220 유전자는 24 hpf 제브라피쉬의 cDNA 라이브러리에서 분리되었고, pGEM-T 이지 벡터 시스템 (pGEM-T Easy Vector System, Promega, A1360)으로 클로닝 하였다. PCR에 이용된 프라이머는 표 2와 같다: The zebrafish RNF220 gene was isolated from a 24 hpf zebrafish cDNA library and cloned using the pGEM-T Easy Vector System (pGEM-T Easy Vector System, Promega, A1360). The primers used for PCR are shown in Table 2:
<2-2> RNF220 유전자의 시간적 및 공간적 발현양상 확인<2-2> Confirmation of temporal and spatial expression patterns of RNF220 gene
RNF220 유전자의 시간적 및 공간적 발현양상을 확인하기 위하여, 홀-마운트 정위치 교잡법(whole-mount in situ hybridization)을 수행하여 확인하였다.To confirm the temporal and spatial expression patterns of the RNF220 gene, hall-mount It was confirmed by performing whole-mount in situ hybridization.
구체적으로, RNF220의 안티센스-디그옥시제닌 표지 RNA 프로브(Antisense digoxigenin-labeled RNA probes)를 DIG 및 형광 RNA 라벨링 믹스(Fluorescein RNA Labeling Mix,Roche catalogue number 11277073910, 11685619910)를 이용하여 제조사 프로토콜에 따라 합성하였다. 광학현미경을 통해 수정 후, 단계별로 적절한 시기의 수정란을 4% 파라포름알데하이드/PBT에 첨가하여 4℃에서 12시간 이상 고정시켰다. 수정 20시간 이상이 지난 수정란은 핀셋으로 융막(chorion)을 제거한 후 꼬리가 휘지 않도록 고정시켰고, 그 이전의 수정란은 고정을 미리 한 후 융막을 제거하였다. 또한, 꼬리 싹(tail bud, 수정 후 10시간)시기부터 0.2 mM PTU(phenylthiourea)/embryonic water를 넣어 피부색소 형성(pigmentation formation)을 억제시켰다. 융막이 제거되고 고정된 수정란은 PBT(1X phosphate buffered saline, 0.1% Tween-20)로 5분 동안 3회 수세한 후, 100% 메탄올(MeOH)을 사용하여 3회 교체해 주었고, 메탄올 상태로 -20℃에서 보관하였다. 메탄올로 보관된 상기 발생배들을 75%, 50% 및 25% MeOH/PBS로 5분씩 단계별로 세척한 뒤, PBT를 사용하여 5분 간격으로 5번 세척하였다. 발생단계에 따라 10 ㎍/㎖ 프로테이나아제 K(proteinase K)를 포함하는 PBT를 상기 발생배에 20분 처리한 뒤, 4% 파라포름알데하이드/PBT로 20분 상온에서 고정시켰다. 발생배가 상하지 않도록 조심히 PBT로 5분 간격으로 5회 세척한 뒤, 300 ㎕ HYB(50% 포름아미드, 5X SSC, 0.1 % Tween-20)를 첨가하고 70℃에서 15분 동안 방치하였다. 그런 다음, 50% HYB(50% 포름아미드, 5X SSC, 5 ㎎/㎖ 토룰라(torula) RNA, 50 ㎍/㎖ 헤파린(heparin), 구연산(Citric acid), pH6, 0.1% Tween-20)을 300 ㎕씩 첨가하고 70℃에서 1 내지 2시간 이상 prehybridization시킨 후 프로브(probe)를 30 내지 100 ng정도 첨가하여 70℃에서 12시간 동안 교배(hybridization)시켰다. 70℃를 유지한 상태에서 프로브(probe)를 제거하고 100%, 75%, 50% 및 25%의 HYB*/0.2X SSCT를 순차적으로 15분마다 세척하였다. 상기와 동일한 온도에서 2 X SSCT로 교체하여 20분 동안 세척하였으며, 0.2X SSCT에서 30분마다 5회 수세하여 하이브리드(hybrid)를 하지 못한 프로브(probe)를 제거하였다. 그런 다음, 상온에서 75%, 50% 및 25%의 0.2X SSCT/PBT를 사용하여 5분 간격으로 순차적으로 교체하고 PBT로 5분마다 5회 수세하였다. 발색 및 발현부위를 확인하기 위하여, 5% 말혈청(horse serum)/PBT를 300 ㎕ 첨가하여 상온에서 1시간 동안 방치하고, 프로브(probe)가 붙지 않은 곳에 혈청(serum)이 붙어 항체반응 시 발색 및 발현이 되지 않게 차단하였다. 그런 다음, 5% 말혈청/PBT에 1/4000배로 희석시킨 항-DIG-AP Fab(anti-DIG-AP Fab)(150 u/200 ㎕; 로슈, 독일)을 300 ㎕ 첨가하고, 상온에서 4시간 또는 4℃에서 12 내지 16시간 동안 반응시켰다. PBT로 10분씩 12번 이상 세척한 다음 염색 버퍼(staining buffer)(0.1 M Tris-Cl pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 0.1% Tween-20)를 사용하여 5분간 3번 세척하였다. 발색기질인 NBT/BCIP(Nitro Blue Tetrazolium/5-Bromo 4-Chloro 3- Indolyl Phosphate)의 저장액(stock)을 염색 버퍼에 각각 4.5 ㎕/㎖(저장액: 디메틸포름아미드 50 ㎎/㎖ NBT를 포함하는 디메틸포름아미드)과 3.5 ㎕/㎖(저장액: 50 ㎎/㎖ BCIP를 포함하는 디메틸포름아미드)를 첨가하여 빛을 차단한 상온에서 반응시킨 후, 현미경으로 관찰하여 염색부위를 확인하였다. 염색이 끝난 수정란들은 정지 용액(stop solution)(1X PBS pH 5.5, 1mM EDTA, 0.1% Tween-20)을 이용하여 5분씩 4번 세척한 다음 100% 메탄올로 10분 동안 4 내지 5번 정도 씻어주어 탈수(dehydration) 시킨 후 -20℃에서 보관하였다. 사진 작업은 100% 글리세롤(glycerol) 또는 벤질 벤조산(Benzyl Benzoate):벤질 알코올(Benzyl Alcohol)(2:1) 용액에서 MZ-16 현미경(라이카, 미국)을 이용하여 수행하였다.Specifically, antisense digoxigenin-labeled RNA probes of RNF220 were synthesized using DIG and fluorescent RNA labeling mix (Fluorescein RNA Labeling Mix, Roche catalog number 11277073910, 11685619910) according to the manufacturer's protocol. . After fertilization through an optical microscope, fertilized eggs at an appropriate time step by step were added to 4% paraformaldehyde/PBT and fixed at 4° C. for more than 12 hours. After 20 hours of fertilization, the chorion was removed with tweezers, and the chorion was fixed so that the tail did not bend. In addition, 0.2 mM PTU (phenylthiourea)/embryonic water was added from the tail bud (10 hours after fertilization) period to suppress pigmentation formation. After the membrane was removed and the fixed fertilized egg was washed 3 times for 5 minutes with PBT (1X phosphate buffered saline, 0.1% Tween-20), it was replaced 3 times using 100% methanol (MeOH), and in methanol state -20 Stored at ℃. The developing embryos stored in methanol were washed step by step with 75%, 50% and 25% MeOH/PBS for 5 minutes each, and then washed 5 times with PBT at intervals of 5 minutes. Depending on the stage of development, PBT containing 10 μg/ml proteinase K was treated in the development embryo for 20 minutes, and then fixed with 4% paraformaldehyde/PBT for 20 minutes at room temperature. After washing 5 times with PBT at intervals of 5 minutes carefully not to damage the embryo, 300 μl HYB (50% formamide, 5X SSC, 0.1% Tween-20) was added and left at 70° C. for 15 minutes. Then, 50% HYB (50% formamide, 5X SSC, 5 mg/ml torula RNA, 50 μg/ml heparin, citric acid, pH6, 0.1% Tween-20) was added to 300 After adding each μl and prehybridization at 70° C. for 1 to 2 hours or more, 30 to 100 ng of the probe was added, followed by hybridization at 70° C. for 12 hours. The probe was removed while maintaining 70° C., and 100%, 75%, 50% and 25% of HYB * /0.2X SSCT were sequentially washed every 15 minutes. At the same temperature as above, it was replaced with 2X SSCT and washed for 20 minutes, and washed with water 5 times every 30 minutes in 0.2X SSCT to remove a probe that failed to hybridize. Then, at room temperature, using 0.2X SSCT/PBT of 75%, 50%, and 25% was sequentially replaced at 5-minute intervals and washed with PBT 5 times every 5 minutes. In order to confirm the color development and expression site, 300 μl of 5% horse serum/PBT is added and left at room temperature for 1 hour, and the color is developed during antibody reaction by attaching serum to the place where the probe is not attached. and expression was blocked. Then, 300 μl of anti-DIG-AP Fab (150 u/200 μl; Roche, Germany) diluted 1/4000-fold in 5% horse serum/PBT was added, and 4 at room temperature hours or at 4° C. for 12 to 16 hours. After washing at least 12 times with PBT for 10 minutes, staining buffer (0.1 M Tris-Cl pH 9.5, 0.1 M NaCl, 50 mM MgCl 2 , 0.1% Tween-20) was used for washing 3 times for 5 minutes. A stock solution of NBT/BCIP (Nitro Blue Tetrazolium/5-Bromo 4-Chloro 3-Indolyl Phosphate), which is a color development substrate, was added to the staining buffer at 4.5 μl/ml (stock solution: dimethylformamide 50 mg/ml NBT). containing dimethylformamide) and 3.5 μl/ml (stock solution: dimethylformamide containing 50 mg/ml BCIP) were added and reacted at room temperature to block light, and then the stained area was confirmed by observation under a microscope. After staining, the fertilized eggs are washed 4 times for 5 minutes each using a stop solution (1X PBS pH 5.5, 1 mM EDTA, 0.1% Tween-20), and then washed 4 to 5 times for 10 minutes with 100% methanol. After dehydration, it was stored at -20°C. Photography was performed using an MZ-16 microscope (Leica, USA) in 100% glycerol or benzyl benzoate:benzyl alcohol (2:1) solution.
그 결과, 도 2에 나타난 바와 같이, rnf220a 전사는 주로 발달 중인 신경계에서 발현하는 것을 확인하였다 (도2).As a result, as shown in FIG. 2 , it was confirmed that rnf220a transcription was mainly expressed in the developing nervous system ( FIG. 2 ).
<2-3> 홀-마운트 면역조직화학 염색법<2-3> Hall-mount immunohistochemical staining method
RNF220 유전자 녹아웃 제브라피쉬의 운동뉴런 및 축삭돌기, 세포죽음, 근육의 형성을 관찰하기 위하여 홀-마운트 면역조직화학 염색법(Whole-mount immunohistochemistry staining)을 수행하였다.Whole-mount immunohistochemistry staining was performed to observe motor neurons, axons, cell death, and muscle formation of RNF220 gene knockout zebrafish.
구체적으로, 수정 후 24시간 및 36시간의 정상 및 RNF220 유전자 녹아웃 제브라피쉬 수정란은 핀셋으로 융막(chorion)을 제거한 후 꼬리가 휘지 않도록 4% 파라포름알데하이드/PBT에 첨가하여 4℃에서 12시간 이상 고정시켰다. 융막이 제거되고 고정된 수정란은 PBT(1X phosphate buffered saline, 0.1% Tween-20)로 5분 동안 3회 수세한 후, 100% 메탄올(MeOH)을 사용하여 3 내지 4번 정도 교체해 주었고, 메탄올 상태로 -20℃에서 보관하였다. 메탄올로 보관된 상기 발생배들을 75%, 50% 및 25% MeOH/PBS로 5분씩 단계별로 세척한 뒤, PBT를 사용하여 5분 간격으로 5번 세척하였다. 2% 말혈청(horse serum)/PBT를 300 ㎕ 첨가하여 상온에서 1시간 동안 방치한 뒤, 2% 말혈청/PBT에 1/500배로 희석시킨 1차 항-Znp-1 마우스 단일클론항체를 300 ㎕, 또는 1/500배로 희석시킨 1차 항-casp3 토끼 단일클론항체 300ul, 또는 1/100배로 희석시킨 1차 항-A4.1025 마우스 단일클론항체 300ul를 첨가하고, 상온에서 4시간 또는 4℃에서 12 내지 16시간 동안 반응시켰다. PBT로 15분간 4번 세척한 뒤, 2%말형청/PBT에 1/600배로 희석시킨 2차 항-마우스-FITC 항체를 500 ㎕ 첨가하고, 상온에서 2시간 동안 반응시켰다. PBT로 15분간 2번 세척한 뒤, 75% 글리세롤(glycerol)에서 MZ-16 현미경(라이카, 미국)을 이용하여 사진작업을 수행하였다.Specifically, normal and RNF220 gene knockout zebrafish fertilized
그 결과, 도 5에 나타난 바와 같이, 정상과 rnf220a 유전자 녹아웃 제브라피쉬의 운동뉴런 및 축삭돌기는 정상적인 발생을 하는 것으로 확인하였고, 세포죽음의 징후를 보이지 않았으며, 근육의 발생도 정상적이었다(도 5).As a result, as shown in FIG. 5 , it was confirmed that the motor neurons and axons of normal and rnf220a gene knockout zebrafish had normal development, showed no signs of cell death, and muscle development was also normal ( FIG. 5 ). ).
<실시예 3> RNF220 유전자 녹아웃 제브라피쉬 라인 제작<Example 3> RNF220 gene knockout zebrafish line production
RNF220 유전자가 녹아웃된 제브라피쉬를 제조하기 위해 CRISPR/Cas9 유전자 가위를 이용하였다. CRISPR/Cas9 gene scissors were used to prepare zebrafish in which the RNF220 gene was knocked out.
구체적으로, RNF220의 12번째 엑손을 타겟팅할 수 있는 가이드 RNA (gRNA)를 크리스퍼스캔 (CRISPRscan)을 이용하여 디자인하였다. Cas9 mRNA는 mMESSAGE mMACHINE T3 전사 키트 (Thermo Fisher Scientific, AM1348)를 이용하여 합성하였고, 염화리튬 (LiCl)를 이용하여 정제하였다. 가이드 RNA와 Cas9 mRNAs를 혼합하여 수정된 배아 세포에 미세주입 (microinjection) 하였다. 12번째 엑손에 돌연변이가 생성되었는지 확인하기 위해, gDNA를 이용한 타겟-특이적 PCR 및 T7 엔도뉴클라아제 I (endonuclease I) 를 이용하였다. gDNA는 50mM NaOH 및 1M Tris-Cl, pH8.0을 이용하여 분리되었다. 구체적으로, gDNA를 주형으로하여 최종 반응 부피 10 ul ~ 20 ul로 PCR 분석을 수행하였다. 하기 표 3의 프라이머로 수행되었으며, 초기 열변성 과정으로 95℃ 3분 1번 반복, 열변성(denature) 과정 95℃ 30초, 결합(annealing) 과정 60℃ 30초, 중합(extension) 과정 72℃ 10초 30번 반복, 최종 중합 과정으로 72℃ 5분 1번 반복하여 PCR을 수행하였다. PCR 반응물의 10 ㎕를 1X TAE 버퍼 및 3.5% 아가로스젤을 이용하여 전기영동하였다. Specifically, a guide RNA (gRNA) capable of targeting the 12th exon of RNF220 was designed using CRISPRscan. Cas9 mRNA was synthesized using the mMESSAGE mMACHINE T3 transcription kit (Thermo Fisher Scientific, AM1348) and purified using lithium chloride (LiCl). Guide RNA and Cas9 mRNAs were mixed and microinjected into fertilized embryonic cells. To confirm whether the mutation was generated in the 12th exon, target-specific PCR using gDNA and T7 endonuclease I (endonuclease I) were used. gDNA was isolated using 50 mM NaOH and 1 M Tris-Cl, pH 8.0. Specifically, PCR analysis was performed with a final reaction volume of 10 ul to 20 ul using gDNA as a template. It was performed with the primers of Table 3 below, the initial heat denaturation process at 95 ° C. for 3 minutes repeated once, the heat denaturation process at 95 ° C. for 30 seconds, the annealing process at 60 ° C. for 30 seconds, the extension process at 72 ° C. PCR was performed by repeating 30 times for 10 seconds, and repeating once for 5 minutes at 72°C as the final polymerization process. 10 μl of the PCR reaction was electrophoresed using 1X TAE buffer and 3.5% agarose gel.
그 결과, 제작된 제브라피쉬에서 RNF220 유전자가 녹아웃된 것을 확인하였다. RNF220 유전자 녹아웃 제브라피쉬의 형태학적 표현형을 확인하기 위해서 80 hpf와 8 dpf에서 야생형 제브라피쉬와 비교하였다. RNF220 유전자가 녹아웃된 제브라피쉬와 야생형 제브라피쉬의 전반적인 발생과정에서의 외형적이고 형태학적인 차이는 확인할 수 없었다 (도 3).As a result, it was confirmed that the RNF220 gene was knocked out in the prepared zebrafish. To confirm the morphological phenotype of RNF220 gene knockout zebrafish, it was compared with wild-type zebrafish at 80 hpf and 8 dpf. There were no external and morphological differences in the overall development process of the zebrafish in which the RNF220 gene was knocked out and the wild-type zebrafish (FIG. 3).
<실험예 1> RNF220 유전자 녹아웃 제브라피쉬의 운동장애 행동 관찰<Experimental Example 1> Observation of movement disorder behavior of RNF220 gene knockout zebrafish
상기 <실시예 >에서 제작된 RNF220 유전자 녹아웃 제브라피쉬의 행동학적 표현형을 확인하기 위해 행동 실험을 진행하였다. A behavioral experiment was conducted to confirm the behavioral phenotype of the RNF220 gene knockout zebrafish produced in <Example>.
구체적으로, 제브라피쉬는 발생 2일째에 부화를 하고, 발생 5일째에 자유수영과 함께 먹이 습식행동이 가능한데, 도 4에 나타난 바와 같이, 8 dpf RNF220 녹아웃 제브라피쉬에서 야생형의 제브라피쉬와 비교하였을 때, 자유수영이 불가능한 운동장애를 보였으며, 이동한 총 거리도 감소하였음을 확인하였다 (도 4). 안구운동 장애를 관찰하기 위해, 5 dpf 야생형 제브라피쉬와 RNF220 유전자 녹아웃 치어를 아가로오즈젤 틀에 넣어 고정시켰다. 제브라피쉬 치어는 입체현미경 (LEICA, MZ16), 카메라 (LEICA DC300FX) 및 현미경 이미지 소프트웨어 (LEICA, IM50)를 이용하여 관찰하였다. 안구 운동의 녹화와 모니터 디스플레이는 Camtasia Studio software (TechSmith, Version 7.0.0)를 이용하여 녹화하였다. 자유 수영은 위해 8 dpf 제브라피쉬는 콘포칼(confocal) 디쉬에 넣어 관찰하였다. Specifically, zebrafish hatch on the 2nd day of development, and wet feeding behavior is possible with free swimming on the 5th day of development. , showed a movement disorder in which free swimming was impossible, and it was confirmed that the total distance moved also decreased (FIG. 4). To observe eye movement disorders, 5 dpf wild-type zebrafish and RNF220 gene knockout fry were placed in an agarose gel mold and fixed. Zebrafish fry were observed using a stereoscopic microscope (LEICA, MZ16), a camera (LEICA DC300FX) and microscopic image software (LEICA, IM50). The eye movements were recorded and the monitor display was recorded using Camtasia Studio software (TechSmith, Version 7.0.0). For free swimming, 8 dpf zebrafish were placed in a confocal dish and observed.
<실험예 2><Experimental Example 2> RNF220 유전자에 의한 신경세포 관련 유전자의 발현양상 확인 Confirmation of expression pattern of neuron-related genes by RNF220 gene
<2-1> RNF220 유전자 녹아웃 제브라피쉬에서 신경세포 관련 유전자의 발현양상 확인<2-1> Confirmation of expression patterns of neuron-related genes in RNF220 gene knockout zebrafish
상기 <실시예 3>에서 제작된 RNF220 유전자 녹아웃 제브라피쉬에서 RNF220 유전자 손실이 특정 신경세포 발생에 미치는 영향을 확인하기 위하여 mRNA 프로브 (probe)를 사용하여 홀-마운트 정위치 교잡법 (whole-mount in situ hybridization)을 수행하였다. In order to confirm the effect of the loss of the RNF220 gene on the development of specific neurons in the RNF220 gene knockout zebrafish prepared in <Example 3>, a whole-mount in situ hybridization method using an mRNA probe (probe) In situ hybridization) was performed.
구체적으로, isl1, 의 안티센스-디그옥시제닌 표지 RNA 프로브 (Antisense digoxigenin-labeled RNA probes)는 DIG 및 형광 RNA 라벨링 믹스(Fluorescein RNA Labeling Mix, Roche catalogue number 11277073910, 11685619910)를 이용하여 제조사 프로토콜에 따라 합성되었으며, 상기 <실시예 2-2>의 홀-마운트 정위치 교잡법(whole-mount in situ hybridization)과 동일한 방법으로 실험을 수행하였다.Specifically, antisense digoxigenin-labeled RNA probes of isl1 were synthesized according to the manufacturer's protocol using DIG and fluorescent RNA labeling mix (Fluorescein RNA Labeling Mix, Roche catalog number 11277073910, 11685619910). and the experiment was performed in the same manner as the whole-mount in situ hybridization of <Example 2-2>.
또한, RNF220 유전자 녹아웃 제브라피쉬의 운동뉴런 및 축삭돌기의 형성, 세포사멸의 확인, 근육의 형성을 확인하기 위하여 각각 항-znp-1, 항-activated caspase 3, 항-A4.1025 항체를 사용하여 상기 <실시예 >의 홀-마운트 면역조직화학 염색법 (whole-mount immunohistochemistry staining)을 수행하였다.In addition, anti-znp-1,
그 결과, 도 5에서 나타난 바와 같이 운동 뉴런을 표지하는 isl1의 발현은 야생형과 RNF220 녹아웃 제브라피쉬에서 차이가 없었으며, 운동 뉴런의 축살돌기에서 발현하는 단백질인 znp-1의 발현 역시 정상이었다. 또한, 세포사멸과 근육의 형성도 야생형과 RNF220 녹아웃 제브라피쉬 사이에서 차이가 없었다 (도 5). 그러나, 도 6에서 나타난 바와 같이 후뇌와 척수에서 V2a의 인터뉴런을 표지하는 vsx2 및 V2a/b 전구체 (precursor)를 표지하는 vsx1의 발현이 RNF220 녹아웃 제브라피쉬에서 크게 줄어들어 있는 것을 확인하였다. 반면, V0 인터뉴런을 표지하는 evx1과 V1 인터뉴런을 표지하는 en1b의 발현은 야생형과 RNF220 녹아웃 제브라피쉬에서 발현의 차이가 없음을 확인하였다 (도 6). 또한, RNF220 녹아웃 제브라피쉬의 뇌에서 glutamate성 뉴런(Glutamatergic neurons)을 표지하는 slc17a6b과 GABA성 뉴런 (GABAergic neurons)을 표지하는 gad1b의 발현을 관찰하였고, 그 결과 RNF220 녹아웃 제브라피쉬의 후뇌에서 slc17a6b의 발현이 크게 감소한 것을 확인하였다. 따라서, RNF220 녹아웃 제브라피쉬에서는 V2a 인터뉴런의 발생이 특이적으로 결손되어 있음을 확인하였다 (도 7).As a result, as shown in FIG. 5 , the expression of isl1, a marker for motor neurons, did not differ between wild-type and RNF220 knockout zebrafish, and the expression of znp-1, a protein expressed in the axon of motor neurons, was also normal. In addition, there was no difference in apoptosis and muscle formation between wild-type and RNF220 knockout zebrafish (FIG. 5). However, as shown in FIG. 6 , it was confirmed that the expression of vsx2, which labels interneurons of V2a in the hindbrain and spinal cord, and vsx1, which labels the V2a/b precursor was significantly reduced in RNF220 knockout zebrafish. On the other hand, it was confirmed that there was no difference in expression in the wild-type and RNF220 knockout zebrafish in the expression of evx1, which labels the V0 interneuron, and en1b, which labels the V1 interneuron (FIG. 6). In addition, the expression of slc17a6b that labels glutamatergic neurons and gad1b that labels GABAergic neurons in the brain of RNF220 knockout zebrafish was observed. As a result, expression of slc17a6b in the hindbrain of RNF220 knockout zebrafish It was confirmed that this significantly decreased. Therefore, it was confirmed that the generation of V2a interneurons was specifically deleted in RNF220 knockout zebrafish (FIG. 7).
<2-2> RNF220 유전자 녹아웃 제브라피쉬에 인간의 RNF220 유전자를 복원하였을 때, 신경세포 관련 유전자의 발현양상 확인<2-2> RNF220 gene knockout When human RNF220 gene is restored in zebrafish, expression pattern of neuron-related genes is confirmed
RNF220 유전자 녹아웃 제브라피쉬에서 인간의 RNF220 유전자를 복원하였을 때, 변이로 인해 유발되었던 신경 발현의 이상이 회복되는지 확인하기 위해, vsx2 유전자의 발현변화를 확인하였다. When the human RNF220 gene was restored in the RNF220 gene knockout zebrafish, in order to confirm whether the abnormality of neuronal expression induced by the mutation was recovered, the expression change of the vsx2 gene was confirmed.
구체적으로, 사람의 정상형, 그리고 운동장애 환자에서 발견된 점돌연변이 H4Y, Y67C, Q327X, R444H, Q505R, R511G RNF220의 합성 capped mRNAs는 mMESSAGE mMACHINE SP6 전사 키트(Ambion, AM1340)를 통해 주형으로 선형화된 플라스미드 DNA를 이용하여 전사되었다. mRNAs는 추적 염료로 0.2% 페놀레드를 포함하는 0.2 M KCl에 용해되었으며, PV820 Pneumatic PicoPump(WPI)를 이용하여 1 세포 단계 배아에 미세주입되었다.Specifically, the synthetic capped mRNAs of the point mutations H4Y, Y67C, Q327X, R444H, Q505R, R511G RNF220 found in normal human and dyskinesia patients were linearized plasmids as templates using the mMESSAGE mMACHINE SP6 transcription kit (Ambion, AM1340). It was transcribed using DNA. mRNAs were dissolved in 0.2 M KCl containing 0.2% phenol red as a tracer dye and microinjected into 1-cell stage embryos using a PV820 Pneumatic PicoPump (WPI).
RNF220
coding
sequence
(서열번호 6)
Human
RNF220
coding
sequence
(SEQ ID NO: 6)
ATGGACTTACACCGGGCAGCCTTCAAGATGGAGAACTCATCCTACCTTCCCAACCCTCTGGCATCCCCAGCACTG
ATGGTCCTGGCATCCACGGCTGAGGCCAGCCGTGATGCTTCCATCCCTTGTCAGCAGCCACGACCCTTTGGTGTAC
CTGTCTCAGTTGACAAGGACGTGCATATTCCTTTCACCAACGGTTCCTATACCTTTGCCTCTATGTACCATCGGCA
AGGTGGGGTGCCAGGCACTTTTGCCAATCGTGATTTCCCCCCTTCTCTACTACACCTCCACCCTCAATTTGCTCCC
CCAAATCTAGATTGCACCCCAATCAGTATGCTGAATCATAGTGGTGTGGGGGCTTTCCGGCCCTTTGCCTCCACCG
AGGACCGGGAGAGCTATCAGTCAGCCTTTACGCCGGCCAAGCGACTTAAGAACTGCCATGACACAGAGTCTCCCCA
CTTGCGCTTCTCAGATGCAGATGGCAAGGAATATGACTTTGGGACACAGCTGCCATCTAGCTCCCCCGGTTCACTA
AAGGTTGATGACACTGGGAAGAAGATTTTTGCTGTCTCTGGCCTCATTTCTGATCGGGAAGCCTCATCTAGCCCAG
AGGATCGGAATGACAGATGTAAGAAGAAAGCAGCGGCATTGTTCGACAGCCAGGCCCCAATTTGCCCCATCTGCCA
GGTCCTGCTGAGGCCCAGTGAGCTGCAGGAGCATATGGAGCAGGAACTGGAGCAGCTAGCCCAACTGCCCTCGAGC
AAGAATTCCCTTCTGAAGGATGCCATGGCTCCAGGCACCCCAAAGTCCCTCCTGTTGTCTGCTTCCATCAAGAGGG
AAGGAGAGTCTCCAACGGCATCACCCCACTCATCTGCCACCGATGACCTCCACCATTCAGACAGATACCAGACCTT
TCTGCGAGTACGAGCCAACCGGCAGACCCGACTGAATGCTCGGATTGGGAAAATGAAACGGAGGAAGCAAGATGAA
GGGCAGAGGGAAGGCTCCTGCATGGCTGAGGATGATGCTGTGGACATCGAGCATGAGAACAACAACCGCTTTGAGG
AGTATGAGTGGTGTGGACAGAAGCGGATACGGGCCACCACTCTCCTGGAAGGTGGCTTCCGAGGCTCTGGCTTCAT
CATGTGCAGCGGCAAAGAGAACCCGGACAGTGATGCTGACTTGGATGTGGATGGGGATGACACTCTGGAGTATGGG
AAGCCACAATACACAGAGGCTGATGTCATCCCCTGCACAGGCGAGGAGCCTGGTGAAGCCAAGGAGAGAGAGGCAC
TTCGGGGCGCAGTCCTAAATGGCGGCCCTCCCAGCACGCGCATCACACCTGAGTTCTCTAAATGGGCCAGTGATGA
GATGCCATCCACCAGCAATGGTGAAAGCAGCAAGCAGGAGGCCATGCAGAAGACCTGCAAGAACAGCGACATCGAG
AAAATCACCGAAGATTCAGCTGTGACCACGTTTGAGGCTCTGAAGGCTCGGGTCAGAGAACTTGAACGGCAGCTAT
CTCGTGGGGACCGTTACAAATGCCTCATCTGCATGGACTCGTACTCGATGCCCCTAACGTCCATCCAGTGTTGGCA
CGTGCACTGCGAGGAGTGCTGGCTGCGGACCCTGGGTGCCAAGAAGCTCTGCCCTCAGTGCAACACGATCACAGCG
CCCGGAGACCTGCGGAGGATCTACTTGTGA
ATGGACTTA C ACCGGGCAGCCTTCAAGATGGAGAACTCATCCTACCTTCCCAACCCTCTGGCATCCCCCAGCACTG
ATGGTCCTGGCATCCACGGCTGAGGCCAGCCGTGATGCTTCCATCCCTTGTCAGCAGCCACGACCCTTTGGTGTAC
CTGTCTCAGTTGACAAGGACGTGCATATTCCTTTCACCAACGGTTCCTATACCTTTGCCTCTATGTACCATCGGCA
AGGTGGGGTGCCAGGCACTTTTGCCAATCGTGATTTCCCCCCTTCTCTACTACACCTCCACCCTCAATTTGCTCCC
CCAAATCTAGATTGCACCCCAATCAGTATGCTGAATCATAGTGGTGTGGGGGCTTTCCGGCCCTTTGCCTCCACCG
AGGACCGGGAGAGCTATCAGTCAGCCTTTACGCCGGCCAAGCGACTTAAGAACTGCCATGACACAGAGTCTCCCCA
CTTGCGCTTCTCAGATGCAGATGGCAAGGAATATGACTTTGGGACACAGCTGCCATCTAGCTCCCCCGGTTCACTA
AAGGTTGATGACACTGGGAAGAAGATTTTTGCTGTCTCTGGCCTCATTTCTGATCGGGAAGCCTCATCTAGCCCAG
AGGATCGGAATGACAGATGTAAGAAGAAAGCAGCGGCATTGTTCGACAGCCAGGCCCCAATTTGCCCCATCTGCCA
GGTCCTGCTGAGGCCCAGTGAGCTGCAGGAGCATATGGAGCAGGAACTGGAGCAGCTAGCCCAACTGCCCTCGAGC
AAGAATTCCCTTCTGAAGGATGCCATGGCTCCAGGCACCCCAAAGTCCCTCCTGTTGTCTGCTTCCATCAAGAGGG
AAGGAGAGTCTCCAACGGCATCACCCCACTCATCTGCCACCGATGACCTCCACCATTCAGACAGATAACCAGACCTT
TCTGCGAGTACGAGCCAACCGGCAGACCCGACTGAATGCTCGGATTGGGAAAATGAAACGGAGGAAGCAAGATGAA
GGGCAGAGGGAAGGCTCCTGCATGGCTGAGGATGATGCTGTGGACATCGAGCATGAGAACAACAACCGCTTTGAGG
AGTATGAGTGGTGTGGACAGAAGCGGATACGGGCCACCACTCTCCTGGAAGGTGGCTTCCGAGGCTCTGGCTTCAT
CATGTGCAGCGGCAAAGAGAACCCGGACAGTGATGCTGACTTGGATGTGGATGGGGATGACACTCTGGAGTATGGG
AAGCCACAATACACAGAGGCTGATGTCATCCCCTGCACAGGCGAGGAGCCTGGTGAAGCCAAGGAGAGAGAGGCAC
TTCGGGGCGCAGTCCTAAATGGCGGCCCTCCCAGCACGCGCATCACACCTGAGTTCTCTAAATGGGCCAGTGATGA
GATGCCATCCACCAGCAATGGTGAAAGCAGCAAGCAGGAGGCCATGCAGAAGACCTGCAAGAACAGCGACATCGAG
AAAATCACCGAAGATTCAGCTGTGACCACGTTTGAGGCTCTGAAGGCTCGGGTCAGAGAACTTGAACGGCAGCTAT
CTCGTGGGGACCGTTACAAATGCCTCATCTGCATGGACTCGTACTCGATGCCCCTAACGTCCATCCAGTGTTGGCA
CGTGCACTGCGAGGAGTGCTGGCTGCGGACCCTGGTGGCAAGAAGCTCTGCCCTCAGTGCAACACGATCACAGCG
CCCGGAGACCTGCGGAGGATCTACTTGTGA
RNF220 point mutations
RNF220 point mutations
그 결과, 도 8에 나타난 바와 같이, 사람의 정상형 RNF220 mRNA를 미세주입한 경우 vsx2의 발현이 회복됨을 확인하였다 (도 8). 하지만, 발달장애, 지적장애, 운동장애, 근위축측삭경화증 (ALS) 환자에서 발견된 점돌연변이가 포함된 H4Y, Y67C, Q327X, R444H, Q505R, R511G 발현벡터의 RNF220 mRNAs를 주입한 경우 H4Y, Q327X, R444H, R511G mRNAs 미세주입에서는 일부 회복이 실패하였으며, Y67C, Q505R 변이 RNF220 mRNAs 미세주입의 경우 vsx2 발현이 정상수준으로 회복됨을 관찰하였다. As a result, as shown in FIG. 8 , it was confirmed that the expression of vsx2 was restored when human normal RNF220 mRNA was microinjected ( FIG. 8 ). However, when RNF220 mRNAs of H4Y, Y67C, Q327X, R444H, Q505R, R511G expression vectors containing point mutations found in patients with developmental disabilities, intellectual disabilities, movement disorders, and amyotrophic lateral sclerosis (ALS) were injected, H4Y, Q327X , It was observed that some recovery failed in microinjection of R444H and R511G mRNAs, and vsx2 expression was restored to normal level in microinjection of Y67C, Q505R mutant RNF220 mRNAs.
위와 같은 결과를 바탕으로 RNF220 녹아웃 제브라피쉬의 운동 장애 관련 질환 모델 동물로서의 활용 및 발달장애, 지적장애, 운동장애, 근위축측삭경화증 (ALS) 환자에서 발견되는 점돌연변이에 대한 생물학적인 검증을 위한 도구로 사용이 가능함을 밝혔다.Based on the above results, the use of RNF220 knockout zebrafish as a movement disorder-related disease model animal and a tool for biological verification of point mutations found in patients with developmental disabilities, intellectual disabilities, movement disorders, and amyotrophic lateral sclerosis (ALS) indicated that it can be used as
<실시예 4> RNF220 유전자 녹아웃 제브라피쉬를 이용한 근위축성 측색 경화증 (ALS) 및 운동성 장애 치료제 후보 약물의 스크리닝 방법<Example 4> Screening method of drug candidate for treatment of amyotrophic lateral sclerosis (ALS) and movement disorder using RNF220 gene knockout zebrafish
<4-1> RNF220 녹아웃 제브라피쉬의 사육 및 발생배의 준비<4-1> Breeding of RNF220 knockout zebrafish and preparation of embryos
제브라피쉬는 상기 <실시예 1>의 제브라피쉬 사육 및 관리 방법에 따라 준비하고, 실험 하루 전날 오후에 RNF220 유전자 녹아웃 제브라피쉬 암수 한 쌍을 전용 메이팅 케이지(mating cage)에 넣어준 후, 암실에서 보관한다. 다음날 오전에 형광등하에서 빛으로 제브라피쉬 암수 한쌍의 체외 수정을 유도하여 수정란을 수집한다. 수집된 수정란은 페트리디쉬로 옮겨 28.5℃의 배양기에서 발생시키고, 수정 후 5일째부터 비정상적인 움직임을 보이는 RNF220 유전자 녹아웃 제브라피쉬를 선별한다.Zebrafish were prepared according to the zebrafish breeding and management method of <Example 1>, and a pair of male and female RNF220 gene knockout zebrafish were placed in a dedicated mating cage on the afternoon of the day before the experiment, and then stored in a dark room. do. In the morning of the next day, in vitro fertilization of a male and female zebrafish pair was induced with light under fluorescent light to collect fertilized eggs. The collected fertilized eggs are transferred to a Petri dish and generated in an incubator at 28.5°C, and RNF220 gene knockout zebrafish showing abnormal movement from the 5th day after fertilization are selected.
<4-2> 근위축성 측색 경화증 (ALS) 및 운동성 장애 치료제 후보 약물의 스크리닝 방법<4-2> Screening method for drug candidates for amyotrophic lateral sclerosis (ALS) and movement disorders
상기 <실시예 4-1>에서 선별된 RNF220 유전자 녹아웃 제브라피쉬를 이용하여, 근위축성 측색 경화증 (ALS) 및 운동성 장애 질환 모델로서 활용이 가능함을 확인하기 위해 후보 약물의 스크리닝 실험과정은 다음과 같다.In order to confirm that the RNF220 gene knockout zebrafish selected in <Example 4-1> can be used as a model for amyotrophic lateral sclerosis (ALS) and movement disorder disease, the screening test procedure of a candidate drug is as follows. .
구체적으로, 수정 후 5일째의 선별된 rnf220a 동형접합 녹아웃 제브라피쉬를 한 마리씩 96웰 플레이트에 넣고, 피검화합물을 다양한 농도로 처리한다. 예를 들어, 식물추출물의 경우 6.25, 12.5, 25, 50, 100, 200 ug/ml의 농도로 각각 약 30분 동안 발생배에 노출시킨 후, 자유수영으로 이동한 거리를 입체현미경(LEICA, MZ16), 카메라(LEICA DC300FX) 및 현미경 이미지 소프트웨어(LEICA, IM50)를 이용하여 관찰하고, Camtasia Studio software(TechSmith, Vesion 7.0.0)를 이용하여 녹화한다. 모든 처리는 대조군과 함께 진행하며, 약물에 대한 음성 대조군으로써 0.5% DMSO를 처리한다. 최종적으로 녹화된 영상을 분석하고, 자유수영을 회복하였는지 확인하여, 피검화합물이 운동장애 개선 및 치료에 효능을 보이는지 확인할 수 있다.Specifically, the selected rnf220a homozygous knockout zebrafish on the 5th day after fertilization are put into a 96-well plate one by one, and the test compound is treated at various concentrations. For example, in the case of plant extracts, after exposure to embryonic embryos at concentrations of 6.25, 12.5, 25, 50, 100, and 200 ug/ml for about 30 minutes, the distance moved to free swimming was measured under a stereoscopic microscope (LEICA, MZ16). ), a camera (LEICA DC300FX) and microscope image software (LEICA, IM50) were used for observation, and the recording was performed using Camtasia Studio software (TechSmith, Vesion 7.0.0). All treatments were run with the control, and 0.5% DMSO was treated as a negative control for the drug. Finally, by analyzing the recorded image and confirming whether free swimming is restored, it is possible to confirm whether the test compound is effective in improving and treating movement disorders.
위와 같은 과정을 통하여 RNF220 유전자 녹아웃 제브라피쉬는 발달장애, 지적장애, 운동장애 질환 관련 약물 스크리닝 방법에 유용하게 사용될 수 있다.Through the above process, the RNF220 gene knockout zebrafish can be usefully used in drug screening methods related to developmental disorders, intellectual disabilities, and movement disorders.
<110> Chungnam National University <120> rnf220a knock-out transgenic animal model and using thereof <130> 2019p-08-019 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1734 <212> DNA <213> Artificial Sequence <220> <223> rnf220a DNA sequence <400> 1 atggacttgc atcgggcggc tttcaaaatg gagagctcct cctacctacc caacccgtta 60 gcatcccctg cactaatggt gctagcatcg acggctgagg ctagccgtga tgcctccatc 120 ccctgccaac agcctcggcc attcggtgtg ccagtgtctg tggagaagga cgttcacctg 180 cccttcaaca atggttccta tacgtttgcc tccatgtacc accgacaggg cgccgtacca 240 ccaggcttcc ccaacaggga ctttcctcct tcccttctac acctccatca tcagtttgca 300 ccacccaatc tggactgctc ccccataagc atgctcaacc acagtggtgt cggggccttc 360 cggccattcg cttctcctcc ggaagatcgt gatggagcgg gtggtggata ccagtctgca 420 ttcacccctg ccaagaggct aaagggctgc ctggaggccg aggcgtcccc gcacttgcgc 480 tactcagacg cagaagggaa agaatatgac tttggtggtg ctcagatccc atccagctca 540 cccagtgctc tcaaagctgt ggaagatgca gggaagaaga tcttcgcagt gtcaggcctg 600 ctgtcagatc gagaaacctc ttccagccca gaggatcgca ttgagcgctg taagaagaaa 660 gtgtcactct atgacagtca ggcacccatc tgccccatct gtcaggttct tttgcgtccg 720 ggcgaactac aggagcacat ggagcaggag atggagaggc tcacccacat gcacatcagc 780 aagaacccat cacacaagga catcaatgtg gctccaggca caccgaagtc tctcctgttg 840 tcagtgcaca tcaaacgtga gggcgagtct ccagttgtgt ccccgctctc ctctgatgaa 900 gctcaccatt ctgacagata ccagacgttt ttacgagtgc gagctaacag gcaaacacga 960 ttgaatgccc ggattgggaa aatgaagcgc cggaagcctg aggatggaca gagggaaggt 1020 gcaacagagg aggattctgc agatgttgaa ggagagaacg gaacgcggtt tgaagagtac 1080 gagtggtgcg gacaaaagag ggtccgagct actacattac tggaaggtgg tttcagagga 1140 acaggctttg ccatgtgtag cacgaaggag agccatgaca gtgacgcaga cctggacgtg 1200 gatggagatg acacactgga gtacggcaaa gcccagtaca ctgaagcaga catcattcct 1260 tgctctggag aggaccaggg agaggccaaa gaacgtgagg cacttcgtgg agccgttttg 1320 aatggtggcg tgccgtccaa tagaataaca cctgagttct ccaagtgggc cagtgatgaa 1380 atgccatcca caagtaatgg cgagagcagc aaacaagagc ccagctcttc atcttcgtct 1440 tccacacaga gaacctgtaa aaacagcgag atagagaaga tcacagagga ctcgacagcg 1500 accacactgg aggcgctaaa ggcccgcata agagaactgg agaagcagat cctcagagga 1560 gaccgataca agtgtctcat atgcatggac tcttatacaa tgccactcac ctccatccaa 1620 tgttggcacg tgcactgtga agaatgctgg ctaaggactc tgggaaacaa gaaactctgc 1680 ccacaatgca acaccatcac gtcaccaggg gacctcaggc gtgtctattt gtga 1734 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rnf220a F <400> 2 atgagttctg ggagttgtgg cg 22 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rnf220a F <400> 3 cagcccggat tcttttagtt tcg 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rnf220a exon12 F <400> 4 gatcacagag gactcgacag c 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rnf220a exon12 R <400> 5 tgagacactt gtatcggtct cc 22 <210> 6 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> Human RNF220 coding sequence <400> 6 atggacttac accgggcagc cttcaagatg gagaactcat cctaccttcc caaccctctg 60 gcatccccag cactgatggt cctggcatcc acggctgagg ccagccgtga tgcttccatc 120 ccttgtcagc agccacgacc ctttggtgta cctgtctcag ttgacaagga cgtgcatatt 180 cctttcacca acggttccta tacctttgcc tctatgtacc atcggcaagg tggggtgcca 240 ggcacttttg ccaatcgtga tttcccccct tctctactac acctccaccc tcaatttgct 300 cccccaaatc tagattgcac cccaatcagt atgctgaatc atagtggtgt gggggctttc 360 cggccctttg cctccaccga ggaccgggag agctatcagt cagcctttac gccggccaag 420 cgacttaaga actgccatga cacagagtct ccccacttgc gcttctcaga tgcagatggc 480 aaggaatatg actttgggac acagctgcca tctagctccc ccggttcact aaaggttgat 540 gacactggga agaagatttt tgctgtctct ggcctcattt ctgatcggga agcctcatct 600 agcccagagg atcggaatga cagatgtaag aagaaagcag cggcattgtt cgacagccag 660 gccccaattt gccccatctg ccaggtcctg ctgaggccca gtgagctgca ggagcatatg 720 gagcaggaac tggagcagct agcccaactg ccctcgagca agaattccct tctgaaggat 780 gccatggctc caggcacccc aaagtccctc ctgttgtctg cttccatcaa gagggaagga 840 gagtctccaa cggcatcacc ccactcatct gccaccgatg acctccacca ttcagacaga 900 taccagacct ttctgcgagt acgagccaac cggcagaccc gactgaatgc tcggattggg 960 aaaatgaaac ggaggaagca agatgaaggg cagagggaag gctcctgcat ggctgaggat 1020 gatgctgtgg acatcgagca tgagaacaac aaccgctttg aggagtatga gtggtgtgga 1080 cagaagcgga tacgggccac cactctcctg gaaggtggct tccgaggctc tggcttcatc 1140 atgtgcagcg gcaaagagaa cccggacagt gatgctgact tggatgtgga tggggatgac 1200 actctggagt atgggaagcc acaatacaca gaggctgatg tcatcccctg cacaggcgag 1260 gagcctggtg aagccaagga gagagaggca cttcggggcg cagtcctaaa tggcggccct 1320 cccagcacgc gcatcacacc tgagttctct aaatgggcca gtgatgagat gccatccacc 1380 agcaatggtg aaagcagcaa gcaggaggcc atgcagaaga cctgcaagaa cagcgacatc 1440 gagaaaatca ccgaagattc agctgtgacc acgtttgagg ctctgaaggc tcgggtcaga 1500 gaacttgaac ggcagctatc tcgtggggac cgttacaaat gcctcatctg catggactcg 1560 tactcgatgc ccctaacgtc catccagtgt tggcacgtgc actgcgagga gtgctggctg 1620 cggaccctgg gtgccaagaa gctctgccct cagtgcaaca cgatcacagc gcccggagac 1680 ctgcggagga tctacttgtg a 1701 <110> Chungnam National University <120> rnf220a knock-out transgenic animal model and using thereof <130> 2019p-08-019 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1734 <212> DNA <213> Artificial Sequence <220> <223> rnf220a DNA sequence <400> 1 atggacttgc atcgggcggc tttcaaaatg gagagctcct cctacctacc caacccgtta 60 gcatcccctg cactaatggt gctagcatcg acggctgagg ctagccgtga tgcctccatc 120 ccctgccaac agcctcggcc attcggtgtg ccagtgtctg tggagaagga cgttcacctg 180 cccttcaaca atggttccta tacgtttgcc tccatgtacc accgacaggg cgccgtacca 240 ccaggcttcc ccaacaggga ctttcctcct tcccttctac acctccatca tcagtttgca 300 ccacccaatc tggactgctc ccccataagc atgctcaacc acagtggtgt cggggccttc 360 cggccattcg cttctcctcc ggaagatcgt gatggagcgg gtggtggata ccagtctgca 420 ttcacccctg ccaagaggct aaagggctgc ctggaggccg aggcgtcccc gcacttgcgc 480 tactcagacg cagaagggaa agaatatgac tttggtggtg ctcagatccc atccagctca 540 cccagtgctc tcaaagctgt ggaagatgca gggaagaaga tcttcgcagt gtcaggcctg 600 ctgtcagatc gagaaacctc ttccagccca gaggatcgca ttgagcgctg taagaagaaa 660 gtgtcactct atgacagtca ggcacccatc tgccccatct gtcaggttct tttgcgtccg 720 ggcgaactac aggagcacat ggagcaggag atggagaggc tcacccacat gcacatcagc 780 aagaacccat cacacaagga catcaatgtg gctccaggca caccgaagtc tctcctgttg 840 tcagtgcaca tcaaacgtga gggcgagtct ccagttgtgt ccccgctctc ctctgatgaa 900 gctcaccatt ctgacagata ccagacgttt ttacgagtgc gagctaacag gcaaacacga 960 ttgaatgccc ggattgggaa aatgaagcgc cggaagcctg aggatggaca gagggaaggt 1020 gcaacagagg aggattctgc agatgttgaa ggagagaacg gaacgcggtt tgaagagtac 1080 gagtggtgcg gacaaaagag ggtccgagct actacattac tggaaggtgg tttcagagga 1140 acaggctttg ccatgtgtag cacgaaggag agccatgaca gtgacgcaga cctggacgtg 1200 gatggagatg acacactgga gtacggcaaa gcccagtaca ctgaagcaga catcattcct 1260 tgctctggag aggaccaggg agaggccaaa gaacgtgagg cacttcgtgg agccgttttg 1320 aatggtggcg tgccgtccaa tagaataaca cctgagttct ccaagtgggc cagtgatgaa 1380 atgccatcca caagtaatgg cgagagcagc aaacaagagc ccagctcttc atcttcgtct 1440 tccacacaga gaacctgtaa aaacagcgag atagagaaga tcacagagga ctcgacagcg 1500 accacactgg aggcgctaaa ggcccgcata agagaactgg agaagcagat cctcagagga 1560 gaccgataca agtgtctcat atgcatggac tcttatacaa tgccactcac ctccatccaa 1620 tgttggcacg tgcactgtga agaatgctgg ctaaggactc tgggaaacaa gaaactctgc 1680 ccacaatgca acaccatcac gtcaccaggg gacctcaggc gtgtctattt gtga 1734 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rnf220a F <400> 2 atgagttctg ggagttgtgg cg 22 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rnf220a F <400> 3 cagccccggat tcttttagtt tcg 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rnf220a exon12 F <400> 4 gatcacagag gactcgacag c 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rnf220a exon12 R <400> 5 tgagacactt gtatcggtct cc 22 <210> 6 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> Human RNF220 coding sequence <400> 6 atggacttac accgggcagc cttcaagatg gagaactcat cctaccttcc caaccctctg 60 gcatccccag cactgatggt cctggcatcc acggctgagg ccagccgtga tgcttccatc 120 ccttgtcagc agccacgacc ctttggtgta cctgtctcag ttgacaagga cgtgcatatt 180 cctttcacca acggttccta tacctttgcc tctatgtacc atcggcaagg tggggtgcca 240 ggcacttttg ccaatcgtga tttcccccct tctctactac acctccaccc tcaatttgct 300 cccccaaatc tagattgcac cccaatcagt atgctgaatc atagtggtgt gggggctttc 360 cggccctttg cctccaccga ggaccgggag agctatcagt cagcctttac gccggccaag 420 cgacttaaga actgccatga cacagagtct ccccacttgc gcttctcaga tgcagatggc 480 aaggaatatg actttgggac acagctgcca tctagctccc ccggttcact aaaggttgat 540 gacactggga agaagatttt tgctgtctct ggcctcattt ctgatcggga agcctcatct 600 agcccagagg atcggaatga cagatgtaag aagaaagcag cggcattgtt cgacagccag 660 gccccaattt gcccccatctg ccaggtcctg ctgaggccca gtgagctgca ggagcatatg 720 gagcaggaac tggagcagct agcccaactg ccctcgagca agaattccct tctgaaggat 780 gccatggctc caggcacccc aaagtccctc ctgttgtctg cttccatcaa gagggaagga 840 gagtctccaa cggcatcacc ccactcatct gccaccgatg acctccacca ttcagacaga 900 taccagacct ttctgcgagt acgagccaac cggcagaccc gactgaatgc tcggattggg 960 aaaatgaaac ggaggaagca agatgaaggg cagagggaag gctcctgcat ggctgaggat 1020 gatgctgtgg acatcgagca tgagaacaac aaccgctttg aggagtatga gtggtgtgga 1080 cagaagcgga tacgggccac cactctcctg gaaggtggct tccgaggctc tggcttcatc 1140 atgtgcagcg gcaaagagaa cccggacagt gatgctgact tggatgtgga tggggatgac 1200 actctggagt atgggaagcc acaatacaca gaggctgatg tcatcccctg cacaggcgag 1260 gagcctggtg aagccaagga gagagaggca cttcggggcg cagtcctaaa tggcggccct 1320 cccagcacgc gcatcacacc tgagttctct aaatgggcca gtgatgagat gccatccacc 1380 agcaatggtg aaagcagcaa gcaggaggcc atgcagaaga cctgcaagaa cagcgacatc 1440 gagaaaatca ccgaagattc agctgtgacc acgtttgagg ctctgaaggc tcgggtcaga 1500 gaacttgaac ggcagctatc tcgtggggac cgttacaaat gcctcatctg catggactcg 1560 tactcgatgc ccctaacgtc catccagtgt tggcacgtgc actgcgagga gtgctggctg 1620 cggaccctgg gtgccaagaa gctctgccct cagtgcaaca cgatcacagc gcccggagac 1680 ctgcggagga tctacttgtg a 1701
Claims (17)
The RNF220 gene represented by SEQ ID NO: 1 is deleted, motor neurons and axons develop normally, V2a interneurons and glutamatergic neurons do not normally occur, GABAergic neurons are Characterized in the normal occurrence, amyotrophic lateral sclerosis (ALS) zebrafish model.
The method according to claim 1, wherein the expression of isl1 (ISL LIM Homeobox 1), which is a marker of a motor neuron, a marker for a motor neuron, is normal, and the expression of znp-1, a protein expressed in the axon of a motor neuron, is normal, and the expression of V2a The expression of vsx2 (Visual System Homeobox 2), a marker for interneurons, and vsx1 (Visual System Homeobox 1), a marker for V2a/b precursors, decreased, and slc17a6b [solute carrier family 17 (sodium) -dependent inorganic phosphate cotransporter), member 6b] is reduced, and the expression of gad1b (glutamate decarboxylase 1b), which labels GABAergic neurons, is normal, a zebrafish model.
According to claim 1, wherein the model is characterized in that the recovery of the interneuron generation of V2a when the RNF220 gene is restored, the zebrafish model.
2) 상기 단계 1)의 콘스트럭트를 수정란에 도입하는 단계;
를 포함하는 것을 특징으로 하는, 제1항의 근위축측삭경화증 제브라피쉬 모델의 제조방법.
1) preparing an RNF220 gene knock-out construct; and
2) introducing the construct of step 1) into the fertilized egg;
A method of manufacturing a zebrafish model of amyotrophic lateral sclerosis of claim 1, characterized in that it comprises a.
2) 상기 단계 1)의 피검화합물이 처리된 제브라피쉬를 무처리된 대조군과 비교하여 증상 회복여부를 판단하는 단계;
를 포함하는 것을 특징으로 하는 근위축측삭경화증 치료용 약물의 스크리닝 방법.
1) treating the test compound in the zebrafish model of claim 1; and
2) comparing the zebrafish treated with the test compound of step 1) with an untreated control group to determine whether symptoms are recovered;
A screening method of a drug for the treatment of amyotrophic lateral sclerosis, comprising a.
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| Molecular Autism 8:50, DOI 10.1186/s13229-017-0168-2(2017년 공개)* |
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