Name: Gait Analysis in an Angelman Syndrome Mouse Model
Uploaded: 2023-10-20T12:10:00
Description: Watch this Scientific Journal Video about Gait Analysis in an Angelman Syndrome Mouse Model at JoVE.com

gait analysis in an angelman syndrome mouse model

행동 테스트를 사용하여 Angelman Syndrome의 Murine 모델 검증

Angelman syndrome (AS) is a rare neurodevelopmental disease. The most common genetic origin of AS is a large deletion of the 15q11-q13 region of the maternally-derived chromosome, which is found in nearly 74% of patients1. Deletion of this region causes the loss of UBE3A, the main causative gene of AS that encodes an E3 ubiquitin ligase. The paternal allele of the UBE3A gene in neurons is silenced in a process known as imprinting. As a consequence, paternal imprinting of the gene allows only maternal expression in the central nervous system (CNS)2. Therefore, UBE3A gene deletion from the maternally-derived chromosome leads to the development of AS symptoms. In humans, AS manifests at around 6 months of age, with developmental retardation that persists throughout all developmental stages and results in severe debilitating symptoms in affected individuals3,4. The core symptoms of the disorder include the deficit of fine and gross motor skills, including jerky ataxic gait, serious speech impairment, and intellectual disability. Approximately 80% of AS patients also suffer from sleep disturbances and epilepsy. To date, the only available treatment are symptomatic drugs, which reduce epileptic seizures and improve sleep quality1. Therefore, the development of robust animal models with reproducible behavioral phenotypes alongside refined phenotyping analysis will be essential to elucidate the pathophysiological mechanisms of the disorder and discover effective medications and treatments.
The complexity of the human disorder affecting the CNS demands model organisms to possess a comparable genome, physiology, and behavior. Mice are popular as a model organism due to their short reproductive cycle, small size, and relative ease of DNA modification. In 1984, Paul Willner proposed three basic disease model validation criteria: the construct, face, and predictive validity, which are used to determine the model&#39;s value5. Simply, construct validity reflects the biological mechanisms responsible for the disorder development, face validity recapitulates its symptoms, and predictive validity describes the model response to therapeutic drugs.
To adhere to the above principles, we have chosen the most common genetic etiology, a large deletion of the maternal 15q11.2-13q locus including the UBE3A gene, to create AS model mice. We used the CRISPR/Cas9 technique to delete a 76,225 bp long region spanning the entire UBE3A gene, encompassing both the coding and non-coding elements of the gene, in mice from a C57BL/6N background6. We then bred the animals to obtain UBE3A+/&#8722; heterozygous mice. For face validation of the model, we used animals from crosses of UBE3A+/&#8722; females and wild-type males to gain UBE3A+/- progeny (strain named C57BL/6NCrl-UBE3A&#60;em1(IMPC)Ccpcz&#62;/Ph and later assigned as UBE3AmGenedel/+) and control littermates. We tested their fine and gross motor skills, emotionality, and affect to recapitulate core AS symptoms. In a previous article, we also evaluated the animals&#39; cognitive functions, as AS patients also suffer from intellectual disability6. However, we found no cognitive impairments in UBE3AmGenedel/+ mice, perhaps due to the young age of the animals at the time of testing7. Later examination of the older animals, around 18 weeks old, revealed a deficit in behavioral flexibility during reversal learning in the place preference paradigm. However, the complexity of the employed equipment for this analysis requires a separate methodological module and it is not included here.
The behavioral tests presented here belong to the common phenotyping tools in genetic research, thanks to their high predictive value and sufficient construct validity8,9,10. We used these tests to validate a mouse model of AS by recapitulating core symptoms of the human disease in a reproducible, age-independent manner. The emotionality of the animal was evaluated in the elevated plus maze and open field tests. Both of these tests are based on the approach-avoidance conflict, where animals explore a new environment in search of food, shelter, or mating opportunities while simultaneously avoiding anxiogenic compartments11. Additionally, the open field test is used to test a mouse&#39;s locomotor activity8. The tail suspension test is widely used in depression research to screen for new antidepressant drugs or depressive-like phenotypes in mouse knockout models12. This test evaluates the despair that animals develop over time in an inescapable situation. Motor learning and detailed gait characteristics were determined on the rotarod and in DigiGait, respectively. Animal endurance on the accelerating rod characterizes its balance and movement coordination skills, while detailed analysis of a mouse&#39;s step patterns is a sensitive evaluation of neuromuscular impairments connected to many neurogenerative movement disorders13,14,15. The nestlet shredding test is part of the standard methodology for detecting impulsive behavior in rodents, and since it utilizes natural rodent building behavior, it indicates the animal&#39;s well-being16,17.
The size of the experimental groups was a result of a compromise to meet the 3R rule demands and efficient usage of colony breeding performance. However, to obtain statistical power, the groups had no fewer than 10 individuals, due to the establishment of a sufficient amount of breeding pairs. Unfortunately, breeding performance did not always result in a sufficient number of animals.

저자 스포트라이트: Angelman Syndrome 쥐 모델을 검증하기 위한 고도로 재현 가능한 행동 테스트 배터리 

Angelman 증후군 마우스 모델의 행동 특성

Our goal is to elucidate the physiological roles of genes using mice as an experimental model. We also focus on developing mouse models of specific diseases that scientists can use to investigate disease causation and develop effective treatments. An example of such activity is the mouse model of Angelman syndrome.Arguably, the most promising new technology with potential is integrating artificial intelligence based on deep learning techniques in animal research. This approach can be used to monitor animal behavior in the home cage directly without need for the experimenter to disturb the animals. The biggest challenge in behavioral studies currently is reproducibility.Therefore, we introduce a battery of tests that validate the Angelman syndrome of model in a way that does not depend on the model's age or species. We use established behavioral tests to validate our AS model. However, with the novel approach for the model development closely resembling the human condition with a large deletion.Using CRISPR technology, we deleted the entire UBE3A gene from the mouse genome. In contrast to other models where the deletion is limited to 3KB. We're working on developing a genetic therapy that could restore a symptoms in a mouse model.

Watch this Scientific Journal Video about Gait Analysis in an Angelman Syndrome Mouse Model at JoVE.com

Gait Analysis in an Angelman Syndrome Mouse Model  

Angelman 증후군 마우스 모델의 보행 분석

트레드밀을 켜고 장치 조명을 켜기 전에 장비 패널에서 벨트 속도를 초당 20cm로 수동으로 설정하십시오. DigiGait Imager 소프트웨어를 실행하고 셔터 속도를 위해 필드에 있는 알비노 마우스의 경우 셔터 속도를 100으로, 필드의 검은색 또는 어두운 마우스의 경우 130으로 설정합니다. 마우스를 트레드밀 벨트에 놓고 약 3초 동안 느린 보행 속도로 설정한 다음 마우스를 계속 관찰하면서 마우스를 멈추고 테스트를 수행할 수 있는지 확인합니다.시작 버튼을 눌러 벨트를 시작하고 10초 동안 녹음합니다. 최소 10-15단계의 명확하고 유창한 운동이 관찰될 수 있는지 확인합니다. 정지 버튼을 눌러 벨트를 멈추고 마우스를 임시 고정 케이지로 되돌립니다.재생을 클릭하고 편집 모드에서 시각적 컨트롤을 사용하여 녹화를 검토하여 유창한 단계로 일련의 이미지에 대한 녹화를 스크리닝합니다. 10-15개의 유창한 움직임을 시작 및 끝 프레임 번호를 관련 필드에 수동으로 작성하여 선택합니다. 동물 ID, 생년월일, 성별, 체중, 벨트 속도, 벨트 각도 등의 동물 정보를 입력합니다.저장을 클릭하여 파일을 저장합니다. 그런 다음 DigiGait Analysis 소프트웨어를 열고 동물 발자국의 비디오 녹화에 대한 완전 자동 분석을 기반으로 보행 분석을 수행합니다. UBE3A 돌연변이 동물의 보행 지수가 변경되었습니다.UBE3A 돌연변이 동물은 더 긴 스윙과 자세를 가졌기 때문에 보폭과 길이가 길어졌습니다. 생쥐의 뒷다리, 추진력, 지속 시간 및 감속도 증가했습니다. 이러한 발견은 또한 피크 스탠스에서 더 큰 기공 면적을 나타냈다.

Research

JoVE Journal

Behavior

Gait Analysis in an Angelman Syndrome Mouse Model

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

연구

행동분석학

Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

Angelman 증후군 마우스 모델의 Elevated Plus Maze Test

Open Field Test in an Angelman Syndrome Mouse Model

Angelman 증후군 마우스 모델의 오픈 필드 테스트

Tail Suspension Test in an Angelman Syndrome Mouse Model

Angelman 증후군 마우스 모델의 꼬리 서스펜션 테스트

Rotarod Test in an Angelman Syndrome Mouse Model

Angelman 증후군 마우스 모델의 Rotarod 테스트

Nestlet Shredding: Nest Building Test in an Angelman Syndrome Mouse Model

네슬렛 파쇄 : Angelman 증후군 마우스 모델의 둥지 건물 테스트