이 작품은 건강의 국립 연구소에 의해 지원되었다 F32 NS078958 (DTB) 및 R01 AG033620 (BG)을 부여합니다.

1. 비행 검사자 조립 <ol><li> 링에 탑승 보안 실린더 체인 클램프를 사용하여 1 대. (접시의 무게를위한 실린더 아래 3cm 정도 둡니다.) (참고 : 우리가 사용하는 항공 실린더는 13.5 cm의 직경 90 ㎝ 길이입니다.) </li><li> 비행 실린더 아래에 미네랄 오일의 얇은 층으로 접시의 무게를 삽입합니다. </li><li> 링에 대한 보안 깔때기 링 클램프와 발톱 클램프를 사용하여 2 대. 깔때기의 바닥 비행 실린더의 윗부분과 같이되도록 깔때기의 높이를 조정합니다. (주 : 깔때기의 선단 직경이 바이알을 통해 떨어지지 않도록 드롭 튜브에 넣고, 튜브의 외경보다 작아야한다.) </li><li> 깔때기의 상단에 드롭 튜브를 삽입하고 발톱 클램프를 사용하여 고정합니다. (참고 : 우리는 25cm 길이의 드롭 튜브를 사용하여 떨어지는 비행 함유 균일 한 힘으로 모든 파리의 일관된 배출 허용이 높이에서 튜브를 드롭 튜브의 내부 직경이 sligh해야한다..바이알 자유롭게 떨어질 수 있도록 병의 외경보다 큰 TLY.) </li><li> 적절한 크기로 폴리 아크릴 시트 (들)을 잘라. (주 : 삽입 시트를 제거 돕기 위해, 폭은 단기 실린더의 내주보다 약간 작아야한다.) </li><li> 시트에 엉 키게 함정의 얇은 층을 적용합니다. 사용하기 전에 1 시간 동안 앉아 수 있습니다. (참고 : 시트의 상단과 하단 삽입 / 제거를 위해 시트를 파악하기 위해 코팅 (약 3cm)에 충분한 공간을 둡니다.) </li><li> 비행 실린더의 폴리 아크릴 아미드 시트를 삽입합니다. </li><li> 소나무지지 브래킷을 사용하여 카메라 트랙을 조립한다. (참고 : 트랙의 바닥이 렌즈를 차단하지 않고 카메라를 지원할 수 있도록 그림 1B를 참조하십시오.). </li><li> 스토퍼를 추가하고 제자리에 나사. (참고 : 장소 카메라가 파노라마 모드에서 전체 플라스틱 시트를 볼 수 있도록 위치에 스토퍼를.) </li></ol> 2. 실험을 실행 <ol> &lt;리&gt; 테스트 할 파리의 병을 수집합니다. 최상의 결과를 위해, 더 이상의 20 파리 / 유리 병을 사용합니다. </li><li> 부드럽게 유리 병의 바닥에 파리를 살짝 뺀 다음 드롭 튜브에 넣고 유리 병을 놓습니다. (참고 :이 좁은 깔때기 구멍을 돌 때까지 유리 병 드롭 튜브를 아래로 떨어지는 병이 깔때기를 돌 때, 파리가 비행 실린더에 분사된다..) </li><li> 빈 유리 병을 제거하는 드롭 튜브를 들어 올립니다. (참고 : 동일한 테스트 그룹의 파리의 여러 병이 하나의 폴리 아크릴 아미드 시트에 정량 할 수있는 우리는 최대 200 파리 (20 파리 각 10 병)가 한 장에 테스트하고 쉽게 이미지가 될 수 있다는 것을 찾을 수 있습니다.. </li><li> 플라스틱 시트를 제거하고 평평한 흰색 표면에 놓습니다. (주 : 벤치 탑은 어두운 색의 경우 흰색 포스터 보드를 사용할 수있다.) </li><li> 플라스틱 시트 위에 카메라 트랙을 조립합니다. 카메라의 시야에있는 시트의 상하 양쪽을받는 시트 위에서 충분히 높아야한다. </li><li> 일을 따라 카메라를 밀어E 트랙 파노라마 이미지를 얻기 위해 &quot;캡처&quot;버튼을 누른 상태. </li><li> 기름에 착륙 비행의 수는 각 시험에 대해 수동으로 계산 될 수있다. </li><li> 반복은 주어진 실험의 모든 조건을 2.2-2.7 단계를 반복합니다. 파리는 각 시험 사이의 시트에서 제거 할 수 있습니다. 대안 적으로, 여러 장의 각 시험 삼아 새로운 시트로 사용될 수있다. </li></ol> 3. 데이터 수집 <ol><li> ImageJ에 소프트웨어를 사용하여 열려있는 이미지 파일. </li><li> 이미지 자르기 만 착지 면적을 포함하는 데 필요한 경우. (이 엉 키게 함정에 코팅 된 영역입니다.) </li><li> 8 비트 그레이 스케일 이미지를 변환합니다. </li><li> 흰색 배경을 필터링 &quot;임계 값&quot;을 만듭니다. (이미지 → → 임계 값을 조정합니다.) </li><li> &quot;입자를 분석&quot;메뉴를 사용하여 각 비행을 식별하는 매개 변수를 설정합니다. 입자를 식별하는 데 사용되는 매개 변수를 정의 (입자 분석 → 분석). 우리는 설정으로, 우리는 발견이를 사용하여5-90 픽셀 2, 0.4 ~ 1.0의 원형의 영역을 정확하게 모든 샘플을 식별합니다. </li><li> 각 입자에 대한 좌표의 생성 된 목록을 사용하여 각 비행의 위치를​​ 측정한다. 픽셀 X 좌표 착지 높이를 계산하기 위해 센티미터로 전환시킬 수있다. </li><li> (Microsoft Excel과 같은) 스프레드 시트로 테이블을 가져옵니다. </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

저자는 공개 할 관심의 충돌이 없습니다.

여기에 기술 된 방법을 사용하여, 우리는 가능한 빠르게 이전보다 더 큰 효율성을 제공 초파리 돌연변이 다수의 비행 성능을 평가할 수 있었다. 우리의 실험을 위해, 우리는 일상적으로 남성과 여성을 분리하고 날개에 손상을 줄 수 침략을 제한하는 낮은 밀도 (20 명 미만 파리 / 유리 병)에 그들을 올립니다. 또 다른 중요한 고려 사항은 유전 적 배경의 차이로 인해 비행 성능의 차이를 적절?...

초파리는 오랜 동작 하나의 유전 적 기초를 연구하는 데 사용되었으며, 연구자 동작 2-6의 각종 유형을 분석 할 수있는 여러 가지 방법을 고안했다. 파리는 신경 근육 질환 (7)의 유용한 모델을 제공하는 유용하고있다. 전위의 행동을 연구하는 데 사용되는 일반적인 분석은 비행 성능입니다. 원래 비행 시험기 비행 결함이있는 돌연변이 체를 식별하기위한 비행 능력 (1)의 정량적 평가에 유용하지만, 높은 처리량 화면에 대한 응용 프로그램을 제한하는 몇 가지 단점이 있습니다 : 오일 코팅 실린더의 사용은 지저분하고 복잡와 같은 특정 기능을 가변 힘으로 튜브에 실린더와 파리의 도입의 길이는 양적 정확성을 감소하고, 그것이 테스터의 라이브 파리를 복구하는 것은 어렵다. 이러한 한계를 극복하기 위해, 우리는 개선의 번호를 포함하는 비행 시험기를 수정했습니다. 우리는 &quot;드롭 TU 추가우리는 쉽게 정리 및 개별 파리의 복구를 위해 수있는 접착제로 코팅 된 이동식 아크릴 시트를 사용하여 실험과 사용자 간의 가변성을 제거하기 위해 파리를 소개하는 &quot;수.. 우리는 양적 정확성과 신뢰성을 개선하기 위해 비행 튜브의 길이를 증가하고있다. 마지막으로 우리는 디지털 카메라와 파리의 방문 높이를 계산하는 이미징 소프트웨어를 사용합니다. 우리는 이러한 개선은 비행 성능에 결함이 대규모 유전 화면을 수행에 관심있는 모든 실험실에 유용 할 것으로 판단된다.

<table border="0" cellpadding="0" cellspacing="0" width="704">
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	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Putty knife</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">630147</td>
			<td style="width:185px;"><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Pine back band moulding ( x2 )</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">156469</td>
			<td><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Furring Strip Board</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">164704</td>
			<td><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Tangle Trap Insect Trap Coating</td>
			<td style="width:171px;">BioControl Network</td>
			<td style="width:132px;">268941</td>
			<td><a href="http://www.biconet.com">www.biconet.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Laptop Computer </td>
			<td style="width:171px;">Apple</td>
			<td style="width:132px;">_</td>
			<td><a href="http://www.apple.com/mac/">www.apple.com/mac/</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">mineral oil</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">BP26291</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">white poster board</td>
			<td style="width:171px;">Staples</td>
			<td style="width:132px;">247403</td>
			<td><a href="http://www.staples.com">www.staples.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polystyrene weighing dish</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S67091A</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ImageJ Software</td>
			<td style="width:171px;">National Institutes of Health</td>
			<td style="width:132px;">_</td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">digital camera</td>
			<td style="width:171px;">Sony</td>
			<td style="width:132px;">DSC-TX7</td>
			<td style="width:185px;"><a href="http://www.store.sony.com">www.store.sony.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">fine forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:132px;">_</td>
			<td><a href="http://www.finescience.com">www.finescience.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polycarbonate cylinder (drop tube)</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">8585K62</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">flight cylinder (acrylic)</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">8486K943</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polycarbonate sheets</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">85585K25</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ring stand (x2)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S47808</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ring support</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S47791</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Three-prong extension clamps (x2)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">05-769-7Q</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">funnel</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">10-500-3</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">chain clamps (x2)</td>
			<td style="width:171px;">VWR</td>
			<td style="width:132px;">21573-275</td>
			<td><a href="http://www.vwr.com">www.vwr.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">glass vials</td>
			<td style="width:171px;">VWR</td>
			<td style="width:132px;">66020-198</td>
			<td><a href="http://www.vwr.com/">www.vwr.com</a></td>
		</tr>
	</tbody>
</table>

an improved method for accurate and rapid measurement of flight performance in drosophila

초파리의 비행 등의 행동 분석을위한 유용한 모델 시스템으로 입증되었습니다. 초기 비행 시험기 관련 낙하는에 날아 오일 코팅 실린더를 졸업, 착륙 높이 실린더의 벽과 접촉 충분한 추진력을 제작하기 전에 지금까지 파리 가을 방법을 평가하여 비행 성능의 측정 기준을 제공했다. 여기에서 우리는 네 개의 주요 개선 비행 시험기의 업데이트 된 버전을 설명합니다. 첫째, 우리는 파리가 사용자들 사이 변동성을 제거 시험과 비슷한 속도로 비행 실린더를 입력 할 수 있도록 &quot;드롭 튜브&quot;를 추가했다. 둘째, 우리는 엉 키게 트랩, 라이브 곤충을 포착하기 위해 설계된 접착제 코팅 이동식 플라스틱 시트에 오일 코팅을 대체했다. 셋째, 우리는 비행 능력을보다 정확하게 식별 할 수 있도록 더 긴 실린더를 사용합니다. 넷째 우리는 비행 성능의 점수를 자동화하는 디지털 카메라와 이미징 소프트웨어를 사용합니다. 이러한 개선은 랩을 허용ID, 비행 행동의 양적 평가, 대규모 데이터 세트와 대규모 유전 화면에 유용합니다.

<html> 그림 1A는 업데이트 된 비행 시험기 어셈블리의 개략도를 보여줍니다. 그림 1B 트랙 디자인은 카메라의 시야를 차단하지 않고 파노라마 이미지를 촬영 할 수 있습니다 보여줍니다. 대표 결과 결함 8-10 알려진 비행이 굼벵이 돌연변이 체의 비행 성능, 야생 형 캔톤-S는 파리에 비교 그림 2에 나타내었다. 제어는 지속적으로 개인 간의 ?...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

여기에서 우리는 높은 처리량 검사를 가능하게 초파리의 비행 성능의 신속하고 정확한 측정하는 방법을 설명합니다.

비행 성능의 정확하고 빠른 측정을위한 개선 된 방법에<em&gt; 초파리</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

11.6K 조회수.  University of Wisconsin-Madison.  여기에서 우리는 높은 처리량 검사를 가능하게 초파리의 비행 성능의 신속하고 정확한 측정하는 방법을 설명합니다. 

비디오: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Drosophila의 유충에서 냄새 기반 행동의 고해상도 측정

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Drosophila에서 Visual과 강한 중재 플라이트 컨트롤을 맡은 이후로 자기 테더 시스템

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Drosophila의 비행 중 시각 인한 악취 추적

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

로블롤리 파인로부터 RNA 분리의 개선 방법 ( P. taeda L.) 및 기타 코니퍼 종

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Drosophila melanogaster를위한 지구력 훈련 프로토콜과 세로 성능 Assays

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

엑소 정량화 및 크기 측정을위한 혁신적인 방법

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...