건강 연구의 캐나다 획 네트워크, 의료 연구를위한 앨버타 헤리티지 재단의 캐나다 연구소에서 교부금에 의해 지원됩니다.

Rotorod 워킹 장치 rotorod는 회전 실린더, 직경 4cm, 35cm 지상 고정하고 투명한 플렉시로 동봉되었습니다. rotorod 실린더는 견인을 용이하게 질감 고무 코팅,으로 덮여있었다. 장치의 바닥은 동물이 떨어졌다 때 부상을 방지하기 위해 거품 층으로 덮여있다. 소형 전기 모터는 고무 벨트를 통해 rotorod을 돌려 전원을 제공했습니다. rotorod 한때 매 5 S. 회전하도록 설정되었습니다 비디오 레코딩 및 운동학 고속 디지털 비디오 카메라는 두 번째의 1000 셔터 속도를 사용하여 동물을 영화에 사용되었다. 동물 rotorod 걸어 동안 전면, 측면 및 후면보기 포착되었습니다. 두 팔 니콘, 미이 차가운 광원과 광섬유 조명 한 세트는 녹음하는 동안 충분한 조명을 제공하기 위해 사용되었습니다. 테이프 소니 DSR 20 DV 캠 데크를 사용하여 프레임별로 프레임을 분석했다. 비디오 프레임은 프레임 그래버와 Macintosh 컴퓨터를 사용하여 점령했다. 절차 및 분석 테스트 6 - OHDA 및 제어 쥐의 걷는 동작의 차이를 평가하기 위해, 이후의 수술 일 15 영상 기록 - 30 (장애로부터 복구가 대부분 완료 예정있을 이후) 두 개의 독립적인 검사를했다 실험 조건에 눈이 관측. nigrostriatal 6 - OHDA의 병변은 왼쪽에 뒷다리 이물과 사지에 지속적인 장애의 결과로, 오른쪽 반구에서 유도되었다. 평가 척도가 평가 쥐 &#39;자세를하고 앞발의 스테핑 움직임으로 개발되었습니다 (Whishaw 외., 2003). 스테핑의 다섯 구성 요소는 평가했다 : <ol><li> 자세는 자세의 등급은 드럼에 쥐의 위치를 조사하여 만들어진; </li><li> 리프트 및 릴리스, 앞발이 드럼에서 채취한어진 스테핑주기의 부분; </li><li> , 쥐의 앞으로 이동으로 사지가 드럼 위에 개최되는 스테핑주기의 부분을 가지고; </li><li> 사전, 사지가 드럼과의 접촉을 회복하기 위해 확장되었다하는 접근의 부분, 그리고 </li><li> 앞발이 드럼에 다시 배치어진 배치. </li></ol> 다음과 같은 기준은 정상적인 움직임을 정의하는 데 사용되었다 : <ul><li> 자세 : 암흑의 시체가 개최되는 머리와 드럼을 통해 지향했다. </li><li> 리프트 및 발매일 : 어깨, 팔꿈치와 손목이 드럼의 표면에서 앞발을 높이는 근육이 수축하고 있습니다. </li><li> 캐리 : 포는 드럼의 표면 위에 개최, 자리가 느슨하게 근육이 수축하고, 자리 팁은 신체의 정중선을 중심으로하고 있습니다. 사전 : 사지가 숫자가 확대와 개방 위치로 semiflexed 위치에서 이동과 앞으로 연장됩니다. </li><li> 위치 : 사지는 어깨, 팔꿈치, 그리고 손목에 확장하고 자리는 쥐의 약 중심에 위치한 자리 2 (정중선에서 자리 초)와 순방향 및 드럼의 표면에 앞발을 장소로 열립니다 몸. </li></ul> 각 운동은 3 점 만점에 평점되었습니다 : 1 = / 운동 = / 움직임이 약간 이상 등장 제어 쥐, 0.5의 닮은, 0 = / 운동 (Whishaw 외, 2003). 명확 이상했습니다. 이 등급의 규모 그리고 5 스테핑 수술후 일 각 쥐에서 운동 1, 3, 5, 7, 9, 15을 평가하는 데 사용되었다. 다섯 조치의 점수는 매일 각 밀고에 대한 평균과 결과가 반복 조치로 일 ANOVA를 받게되었습니다. 6 - OHDA 및 제어 쥐에서 도보 rotorod의 개요 운동 등급 규모는 질적 운동 분석을위한 수술 후 테스트 세션에 적용되었다. 테스트 세션 동안 6 OHDA의 병변 그룹은 통제 그룹, F (1,8) = 33.2, P &lt;0.001했던보다 훨씬 낮은 점수를 받았지만, 테스트 데이에 대한 중요한 영향이 없었 않으며, 거기가 큰 그룹되었습니다 테스트 데이 상호 작용에 의해. 스테핑의 점수는 다음 따로 변수 (; 제어 쥐 들어, 라이트 대 왼쪽 앞발 6 - OHDA의 쥐 병변에 ipsilateral forelimb 비해 병변에 contralateral forelimb)로 forelimb와 함께 분석했다. Forelimb, F (1,40) = 15.8, P &lt;0.01의 큰 효과가 있었고, Forelimb, F (1,40) = 15.0, P &lt;0.01로 그룹의 중요한 상호 작용이있었습니다. 후속 T - 테스트는 6 OHDA 그룹의 contralateral - 투 - 병변 forelimb은 ipsilateral - 투 - 병변 forelimb이나 다를하지 않은 제어 쥐의 앞발보다 훨씬 낮은 점수를 받았다고 지적했다. 운동의 구성 요소가 6 OHDA 그룹에 의해 얻은 가난한 점수, 자세의 대책에 기여하는 결정하기 위해그리고 6 - OHDA 그룹의 ipsilateral와 contralateral 앞발의 스테핑주기의 개별 구성 요소는 컨트롤 쥐에서 얻은 값을 비교했다. 6 - OHDA 그룹은 자세와 contralateral 사지에 의해 만들어진 스테핑 구성 요소의 모든마다 있지만, ipsilateral 사지의 구성 요소 없음 (P는 &lt;0.05). 제어 및 6 - OHDA의 쥐의 자세에 질적 차이 및 설명 제어 쥐, 머리는 수평 위치에서 개최하고, 지원 forelimb와 뒷다리 팔다리가 지원 아치를 형성하는 드럼을 스트래들 수 있습니다. 6 - OHDA 쥐 일반적으로 제어 쥐의 ipsilateral 사지 또는 사지에 대한 발생 이상 최대 드럼 표면에 자신의 장애 가까이 함께 배치 앞발 (뷰어를 향한)과 (와 제어 쥐에 대한 동영상을 볼 수있는 머리를 아래로 걸어 점수 자세에 대해 &quot;1&quot;과 &quot;0&quot;의 점수와 6 OHDA 병변의 동물). 일반적으로 자세의 이상은 쥐의 머리의 상대 위치에서 한 눈에 볼 수 있습니다. Forelimb 스테핑 제어 시퀀스에서 스테핑 forelimb는 어깨, 팔꿈치 및 손목을 flexing에 의해 해제됩니다. 사지가 해제되면서 자리가 느슨하게 근육이 수축하고, 사지가 잘 드럼의 표면 위에 해제됩니다. 사지가 고급 때, 그것은 어깨, 팔꿈치 및 손목에 확장하고이 위치로 자리 연장 및 오픈됩니다. 컨트롤 쥐에 비해, 병변 사지에 contralateral DA - 고갈의 쥐의 모든 구성 요소의 진폭 감쇠됩니다. 사지는 나중에 드럼을 출시 적은을 드러내기 시작하고, 제어 forelimb보다 짧은 거리를 고급 있습니다. 또한, 사지가 해제되면, 자리가 더 근육이 수축되며, 사지가 위치하면 숫자가 적은 제어 쥐보다 확장하고 열립니다. 그것이 드럼의 상단에 도달되면 컨트롤 forelimb이 해제되어 손목이 앞으로 앞발을 들어올려하고 근육이 수축하고, 손목은 사지 진보로 연장됩니다. 반대로, 6 - OHDA 랫은 병변 forelimb에 contralateral의는 드럼의 움직임에 의해 더욱 caudally 실시하고, 따라서 여전히 확장 손목과 나중에 릴리스됩니다. 손목의 굴곡은 수동적으로 나타나고 자리는 드럼의 움직임에 의해 뒤로 실시되며, forelimb가 앞으로 가져되면 발 팔뚝이 드럼 표면에서 명확하게 해제되지 않는 발생합니다. 제어 쥐 forelimb 잘 전달 확장하고 자리는 사지 진보로 엽니다. 사지가 배치되면서, 그것은 숫자 4, 3, 2 연속 다음, 드럼 먼저 표면을 접촉 숫자 5, arpeggio 운동과 함께 배치됩니다. 반대로, 6 - OHDA 랫의 사지가 해제 및 확장 이하의 숫자가 적은을 확장하고 있으며, 그들은 &#39;때려&#39;와 거의 동시에 드럼의 표면을 접촉합니다. 비디오는 &quot;1&quot;로 득점, 주로 &quot;0&quot;으로 득점했다 6 OHDA의 병변 동물의 forelimb 스텝 움직임 있었다 제어 동물에 forelimb 스테핑 움직임을 보여줍니다. 스테핑 뒷다리 6 - OHDA의 쥐의 contralateral - 투 - 병변 뒷다리 사지에 장애도있었습니다. 컨트롤 뒷다리가 뒤로 움직이면서, 그것은 회전 드럼에 대해 밀어 표시하고 그럼으로 발가락이 바깥쪽으로 회전합니다. 사지가 앞으로 움직이면서, 발이 바깥쪽으로 휘둘러됩니다. 6 - OHDA의 쥐 뒷다리는 드럼 회전과 직접 후방 이동하고 뭔가있다면, 약간 안쪽으로 변합니다. 사지가 앞으로 움직이면서, 발 직접 이월됩니다. 제어 랫 스윙 바깥쪽으로하고 선진 한, 발가락이 발목보다 개최의 산기슭과 발가락이 연장. 제어 쥐의 배치, 발가락 다섯 연락처 동안 처음으로 드럼 및 중량의 표면은 발끝과 손바닥에 걸쳐 배포됩니다. 반면, 6 - OHDA 랫의 발가락이 수행하는 동안 근육이 수축하고 배치는 arpeggio 운동없이 이루어집니다. 발가락이 다섯가 먼저 해제하고 발가락 1 마지막으로 해제됩니다으로 제어 쥐의 발가락은 반대 arpeggio 패턴에 출시하고 있습니다. 발목는이 들고 운동하는 동안 자세를 유지 근육이 수축하면서이 운동이 6 - OHDA의 병변 쥐에서 누락되었습니다. 다시 6 OHDA 랫의 사전 움직임이 적은 exorotated 사지와 감쇠 아르 적은 해제 및 제어 쥐보다 적게 진보된. 제어 쥐들이 발가락을 장소로, 그들은 쥐의 본문 5 점 옆으로 상대와 같은 것을 발가락을 연장하고 열 수 있습니다. 토프트 다섯 연락처 먼저 쥐가 발가락 끝부분에 그 무게를 지원하는 것과 같은 나머지 발가락 다음 드럼. 피트가 이월이므로 6 OHDA 랫은 발가락이 약간 근육이 수축되고, 발가락이 발목을 수평 상대하기 위해 낮은 있으며, 기슭은 드럼에 놓여있는 발가락의 덜 개방이 있습니다. 보폭의 길이는 제어 쥐에 비해 다소 짧은 있습니다. 컨트 차이를 계량하기 위해서는rol와 6 - OHDA 뒷다리의 움직임 리프트 및 릴리스 구성 요소, 발전을 수행하고, 배치는 3 점 척도 평가했고 같은 앞발이 있었다. 조치 게시물 외과 20 일 만든 동영상 기록했고, 세 단계를주기는 각 쥐에 대해 평가했다. 큰 그룹의 차이, F (1,8) = 47.6, P &lt;0.001가 발생했습니다. 구성 요소에 후속 T - 테스트는 6 - OHDA의 쥐의 구성 요소가 한보다 제어 쥐의 모든 구성 요소가 상당히 높은 점수를 받았다고 지적했다. 비디오는 &quot;1&quot;로 득점했다 제어 동물의 뒷다리 스테핑 움직임을 보여줍니다. 비디오에 표시되는 6 - OHDA의 움직임을 스테핑 뒷다리는 주로 &quot;0&quot;의 점수로 평가했다.

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Q., Gorny, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arpeggio+and+fractionated+digit+movements+used+in+prehension+by+rats.">Arpeggio and fractionated digit movements used in prehension by rats.</a> Behav Brain Res. 60, 15-24 (1994).</li><li>Whishaw, I. Q., O'Connor, W. T., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+motor+cortex,+nigrostriatal+dopamine+and+caudate-putamen+to+skilled+forelimb+use+in+the+rat.">The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat.</a> Brain. 109, 805-843 (1986).</li><li>Whishaw, I. Q., Pellis, S. M., Gorny, B., Kolb, B., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximal+and+distal+impairments+in+rat+forelimb+use+in+reaching+follow+unilateral+pyramidal+tract+lesions.">Proximal and distal impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions.</a> Behav Brain Res. 56, 59-76 (1993).</li><li>Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+limb+use+by+control+rats+and+unilateral+DA-depleted+rats+in+the+Montoya+staircase+test:+movements,+impairments+and+compensatory+strategies.">Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies.</a> Behav Brain Res. 89, 167-177 (1997).</li></ol>

현재 연구의 목적은 쥐 운동 기술에 대한 rotorod 시험이 질적 자세의 검사, forelimb의 스테핑과 신경계 부상 쥐에서 스텝을 밟고 뒷다리에 사용할 수 있는지 여부를 조사하는 것이었습니다. 분석을 위해, 일방적인 검찰 depletions 및 제어 쥐가 쥐가 앞에 수평 및 사후 전망에서 녹화한 동영상을했다. 자세와 forelimb 동작의 평가 규모는 스테핑 움직임이 만성 다음과 같은 수술을 장애인 있다고 지적했다. 사지 움직임의 ...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

동물이 회전 드럼에 산책하는 rotorod 시험은, 널리 실험실 설치류 동물의 모터 상태를 평가하는 데 사용됩니다. 성능 짐승 드럼 속도의 함수로 드럼에 체류하는 기간에 의해 측정됩니다. 여기서 우리는 작업이 걷는 동작의 질적 측면에 대한 정보의 풍부한 소스를 제공하는보고합니다. 움직임이 고정 위치에서 수행되기 때문에, 그들은 즉시 고속 비디오 녹화 방법을 사용하여 검사하실 수 있습니다. 현재의 연구는 (nigrostriatal 도파민을 고갈에 대한 권리 nigrostriatal 번들로 6 - hydroxydopamine (6 - OHDA)의 주입에 의해 만들어진 헤미 - 파킨슨 아날로그의 쥐의 걷기 운동의 질적 변화를 나타내기 위해 rotorod의 가능성을 검토하기 위해 실시되었다 DA ). 하루를 다음과 같은 수술에서 시작하고 그들이 rotorod를 걸었으로 다음 정기적으로 다음 두 달 동안, 쥐가 정면 측면 및 사후 전망에서 촬영되었다. 문제는 비디오 기록을 프레임별로 프레임 재생에 의해 분석되었다. 동작을 스테핑의 평가 척도는 헤미 - 파긴슨 쥐가 만성 자신의 자세와 DA - 고갈에 contralateral 사지의 사용 장애인다고 지적했다. 단 체위 및 운동 이상을 표시하지 contralateral 팔다리, 그들은 ipsilateral 사지 그랬보다 추진력을 시작하고 유지 이내에 참가했습니다. 이러한 결과뿐만 아니라 DA - 고갈 일방적으로 보조 새로운 적자를 노출뿐만 아니라 rotorod이 운동의 질적 분석을위한 강력한 도구를 제공할 수 보여줍니다.

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

rotorod 시험 헤미 - 파킨슨 아날로그의 쥐의 걷기 운동의 모터 상태를 평가하는 데 사용됩니다.

쥐에서 도보의 질적 분석 방법으로 Rotorod의 사용

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

use-rotorod-as-method-for-qualitative-analysis-walking

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12.9K 조회수.  University of Lethbridge. rotorod 시험 헤미 - 파킨슨 아날로그의 쥐의 걷기 운동의 모터 상태를 평가하는 데 사용됩니다.

비디오: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : 정확한 Genotyping을위한 간단한 방법

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

연구

생물학

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

작업 워킹 레더 렁 : 채점 시스템과 실용적인 프로그램입니다.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

버딩 효모에서 단백질 농도의 함수로 형태학의 표현형의 발전 분석

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

모델 생물체 유전자로 사용하기 위해 Medaka의 태아의 Microinjection

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

미생물 시스템 생물학의 Chemostats의 사용

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

가금류 생산 샘플에서 세균 공동체의 정성 및 정량 평가를위한 하이브리드 DNA 추출 방법

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...