D. 세임스는 G. 해롤드 &amp; 라일라 Y. 자선 재단과 과학의 컬럼비아 대학의 이니셔티브를 매더 감사드립니다. D. 세임스 및 D. Sulzer는 신경 과학 수상의 맥나이트 기술 혁신에 대한 맥나이트 재단 감사 D. Sulzer이니다, NIMH, 그리고 Picower과 파킨슨병의 기초를 주셔서 감사합니다. H. 장 감사는 NARSAD. RH 에드워즈는 마이클 J. 폭스 재단, 국립 파킨슨병 재단니다 그리고 NIMH 감사합니다. 우리는 TIRF 현미경 설치와 기술 지원 6 OHDA 주사와 조언, 유용한 토론, 영상 분석 프로그램에 대한 Merek 시우에 대한 표시 Sonders 및 월 Schmoranzer에 대한 로버트 버크 감사합니다.

이 방법에 보고된 연구에 사용된 <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator 외., 과학 324 (5933)가. 1441년부터 1444년까지 (2009)</a> . 1. 급성 striatal 조각 준비 <ol><li> 급성 striatal 조각을 준비하기 전에, 하나는 산소 인공 뇌척수 (ASCF) 사전 뇌 추출에 적어도 15 분 동안 얼음으로 진정해야합니다. ACSF의 얼음 추위를 유지하고 해부하는 동안 산소. ACSF (MM 년) : NaCl 125, KCl 2.5, NaHCO 3 26, CaCl 2 2.4, MgSO4 1.3, KH 2 PO 4 0.3, 포도당 10, HEPES 5; 산도 7.3-7.4, 290-295 mOsm. </li><li> 마취없이 남성 마우스 목을 벨. </li><li> 마우스의 전체 두뇌를 추출하고, Krazy ® 접착제를 사용하여 vibratome에 게재 vibratome 트레이에 탑재합니다. 브레인 추출 및 설치 2 분 이내에 이루어져야합니다. 이 시간 동안 ACSF 얼음 추위를 유지하고 조직의 건강을 유지하기 위해 산소를해야합니다. </li><li> bregma 1.54 사이에 0.62 1 250μm 두께 코로나 striatal 뇌의 슬라이스 컷. 세 전체 조각은 다음 바늘 6 개 반 조각으로자를 수있는 얻을 것입니다. </li><li> 프로브 로딩하기 전에 최소한 1 시간 동안 실온에서 산소 ACSF의 뇌 조각 품어. 조각은 좋은 FFN511 로딩 표시하고 슬라이스 준비 후 최대 4 시간까지 이미징 대해서는 운영중 상태로 유지됩니다. </li></ol> 2. FFN511를 로딩 <ol><li> FFN511 로딩 솔루션 (ACSF에 10μM FFN511) 준비, 또한 ACSF에 ADVASEP - 7 100μM 준비합니다. 모든 솔루션은 신선한 사전 자른에 로딩하기 위해 적어도 15 분 산소해야합니다. </li><li> 개별적으로 실온에서 30 분 FFN511 로딩 솔루션 슬라이스를 품어. </li><li> 그런 다음 실온 2 30 분 산소 100μM ADVASEP - 7 ACSF에 로드된 슬라이스를 잠복기에 의해 세포 조직 바운드 염료를 제거합니다. 조각은 이제 영상에 대한 준비가되어 있습니다. </li></ol> 3. 뇌 조각의 영상 FFN511 조각에 FFN511의 영상은 multiphoton 레이저 스캐닝 현미경을 사용하여 수행됩니다. 우리는 (우리는 이전 자이스 혈구 LSM 510 NLO multiphoton 레이저 스캐닝 현미경을 사용되었으며 어떤 결과가 자이스 혈구 현미경으로 획득했다) 프레리 울티마 multiphoton 레이저 스캐닝 현미경을 사용하고 있습니다. <ol><li> 레코딩 챔버에서 FFN511 - 로드된 슬라이스 (RC - 27L, 워너)을 놓고 분당 1-2 ML의 흐름 속도와 산소 ACSF superfuse. 그런 다음 실험 기간 동안 움직임을 최소화하기 위해 조각 위에 나일론 스트링과 플래티넘 하프를 놓으십시오. 추가 움직임을 최소화하려면 슬라이스가 이전에 영상으로 챔버에 적어도 10 분 동안 안정 수 있습니다. </li><li> 시각화 및 10X 물 침지 목표 아래 명시야 발광와 지느러미 striatum을 찾습니다. </li><li> 이 지역의 장소와 위치 바이폴라 꼬인 텅스텐 전극 자극 실험을 수행하는 경우. 이상적인 이미징 영역은 자극 양극성 전극의 두 팁 사이에 300 μm의 사이에 위치하고 있습니다. 시야의 중심이 지역에 놓으십시오. </li><li> 이미지 FFN511 -라는 63x (0.9 NA) 물 침지 자외선 목표 아래 striatal 터미널. FFN511는 밴드 패스 필터 (480-520 nm의)를 통해 볼 수 있습니다 스펙트럼 물리 striatal 터미널 최적의 형광에서 마이 타이 레이저 760 nm의에 흥분됩니다. 이미지는 512 X 512 픽셀 해상도로 관심의 75 X 75 μm의 영역을 12 비트 형식으로 캡처됩니다. 자극 destaining 실험은 Z - 비행기 1 μm의로 구분하여 Z 축 이동 5-7 이미지의 Z - 시리즈에 대한 보상은 각 기간 동안 얻을 수 있습니다. </li></ol> 4. FFN511 Destaining FFN511 레이블은 KCl 높은 농도 (우리 70 MM KCl을 사용하여), (+) - 암페타민 황산염 (AMPH, 20 μm의), 또는 전기적 자극에 의​​해 어느 destained 수 있습니다. 에 높은 칼륨의 ACSF 솔루션 두뇌 슬라이스에 적용됩니다 KCl destaining 실험, 2 분 이내 FFN511 레이블 destains. 기록은 XYZ - T의 이미지를 시간의 경과에 FFN511 destaining을 추적할 수 있습니다. 다음은 전기 자극에 의​​존 도파민 터미널 destaining 설명 : <ol><li> 시간이 지남에 따라 FFN511 destaining를 추적하려면, XYZ - T의 이미지가 기록됩니다. 슬라이스의 최소한의 움직임을 (Z 축 미만 4 μm의)되도록하려면없고, Z - 스택의 레이저, 제어 시간 시리즈 이미지로 photobleaching이 자극하기 전에 최소한 5 분 취득해야합니다. 그렇지 않으면, 레이저 파워를 조정합니다. </li><li> 1 주파수, 4 또는 20 Hz에서에서 자극을 시작하면서 이러한 컨트롤 이미지에 따라 XYZ - T의 이미지를 계속합니다. 1, 4 또는 20 Hz에서 (300 μs X 1mA)의 자극은 양극성 전극을 사용하여 마스터 - 8 펄스 발생기 (AMPI, 예루살렘, 이스라엘)을 계기로 ISO - 플렉스 자극 아이 솔 레이터에 의해 로컬 striatum에 적용됩니다. 최소화하기 위해릴리스 사이트의 탈분극의 변화, 우리는 주기적 voltammetry에 의해 결정 최대한의 자극 강도의 150 %를 적용 자극 프로토콜을 사용하고 있습니다. </li><li> 이미지 분석을위한 적절한 소프트웨어를 사용합니다. 우리가 실험실에서, 우리는 번이나로 puncta 형광 및 변경 사항을 수치 IDL의 이미지 J (웨인 Rosband, 건강의 국립 연구소, 락빌, MD) 및 사용자 정의 작성된 소프트웨어 (연구 시스템, 볼더, CO)을 사용합니다. </li></ol> Represenative 결과 그림 1은 striatal 슬라이스에 FFN511로드 지금 완료 보여줍니다. 10X 및 60x 목표를 사용하는 대표 이미지는 Fig.1에 표시됩니다. 도파민 단말기의 선택적 라벨링는 20 μm의의 암페타민에 의해 destained 수 있습니다. 높은 KCl에 의해 FFN511 라벨링의 destaining 그림 2 보여줍니다. 지역 전기적 자극에 의​​해 FFN511 라벨링의 destaining 3 쇼 그림. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="그림 1" /> 그림 1 라이브 피질 - striatal 급성 조각에 FFN511 라벨 도파민 터미널 (A) 급성 라이브 피질 - striatal 통에 FFN511하여 레이블 :.. striatum에서 풍부한 라벨 (STR), 피질에서 sparser 라벨 (CTX), 그리고 노 레이블 코퍼스의 callosum (CC). 스케일 바. 100 μm의 암페타민에 의해 striatum에서 FFN511의 (B) Destaining. 왼쪽 패널 : 전에 암페타민, 오른쪽 패널 : 20 μm의의 암페타민 20 분 후. 스케일 바 : 10 μm의. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="그림 2" /> 그림 2. 높은 KCl에 의해 FFN511 라벨의 Destaining. FFN511 라벨이 ACSF 70 MM KCl 2 분 응용 프로그램 내에서 destained입니다. 스케일 바 : 10μm. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="그림 3" /> 그림 3. striatum에서 FFN511 라벨링의 주파수 의존 destaining가. (A) 4 Hz에서 현지 자극은 터미널에서 destaining 결과. 자극은 t = 0에서 시작되었다. 스케일 바 : 5 μm의 (B) 4 Hz에서에서 FFN511의 Destaining은 칼슘 2 + - 및 주파수 의존.. 컨트롤이 더 자극 (3 조각에서 153 puncta)를받지 않습니다. 카드뮴 염화물 (200 μm의)와 Destaining 것은 unstimulated 컨트롤 (5 조각에서 475 puncta)로 동일했습니다. 로 계산 값은 각각의 자극 주파수에 대한 destaining 곡선은 하나의 지수 감쇠 함수와 반감기 (T 1 / 2)에 의해 맞는 τ X 0.693 (1 Hz에서 : 9 조각, 4 Hz에서에서 765 puncta : 7 조각에서 410 puncta, 20 Hz에서 6 조각)에서 416 puncta. striatal 두뇌 슬라이스 준비 exocytosis 동안 FFN511 destaining의 2 광자 현미경에서 비디오 현미경을 참조하십시오.

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

이 비디오에서는, 우리는 형광 도파민의 analogs를 사용하여 광학 neurotransmission을 시각화하는 방법을 보여줍니다. FFN511 우리가 개발한 FFNs의 첫 세대입니다. 그것은으로 과학에 나타난 시냅스 vesicles로 세포질에서 모노 아민 신경 전달 물질을 실시하고, 특히 피질의 striatum 및 추정 카테 및 / 또는 세로토닌 터미널에서 도파민 터미널 레이블의 연결 기공을 갖는 모노 아민 전송기 (VMAT2)를 (대상으로 설계되었지만 FFN511 상대적으로 소수성이기 때문에 종이), 적절한 로딩 기간은 특이성을 위해 중요합니다. 우리는 그 40 분 이상을위한 인큐베이션은 striatal 조각의 광범위한 특이 현상이 얼룩집니다 발견했습니다. 특이성은 straightforwardly 기능 시냅스 vesicles 이내 FFN의 출시를 일으킬해야 KCl의 높은 농도를 사용 destaining에 의해 결정하실 수 있습니다. 미만 15 분 FFN511하는 슬라이스의 노출은 터미널에있는 색소의 부족 로딩으로 인해 약한 형광 신호가 발생합니다. 이 프로토콜에서는, 우리는 세포 조직 바운드 염료를 제거하는 100μM ADVASEP - 7을 사용합니다. 이 단계는 필요하지 않습니다,하지만 생략하면 ACSF의 워시 아웃 시간 연장해야합니다. 요약, 당신이 선택한 준비에 따라 최적의 레이블을 결정하기 위해 집중 로딩 기간을 수정해야합니다.

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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
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<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
<tr>
<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
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dopamine release at individual presynaptic terminals visualized with ffns

신경계는 시냅스 소포의 융합 동안 신경 전달 물질 방출을 통해 뉴런 사이의 신호를 전송합니다. 신경 전달 물질의 이해와 직접 개별 presynaptic 터미널에서 릴리스를 관찰하기 위해, 우리는 시냅스 소포 모노 아민 전송을위한 기판으로 형광 잘못된 신경 전달 물질을 설계했습니다. striatum의 이미지 도파민 릴리스 이러한 프로브를 사용하여, 우리는 시냅스 소성에 관련 몇 가지 관찰을했다. 우리는 자극마다 신경 전달 물질을 방출하는 시​​냅스 vesicles의 분율이 자극 주파수에 의존 것으로 나타났습니다. kinetically 별개 &quot;예약&quot;시냅스 소포 인구는 이러한 실험 조건 하에서 관찰되지 않았습니다. presynaptic 터미널의 주파수 종속 이질은 그 presynaptic 선택의 주파수 의존 코딩을위한 메커니즘을 나타내는 D2 도파민 수용체에 부분적으로 의존했다 발표했다. 휘 장 및 니코 G.의 Gubernator이 작품에 동등하게 공헌했습니다.

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

새로운 형광 도파민의 analogs를 사용하여 광학 neurotransmission을 측정하는 것을 의미합니다.

개인 Presynaptic 터미널에서 도파민 릴리스 FFNs와 시각

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Nanyang Technological University

Research

JoVE Journal

Biology

24.7K 조회수.  Columbia University. 새로운 형광 도파민의 analogs를 사용하여 광학 neurotransmission을 측정하는 것을 의미합니다.

비디오: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

칼슘 지표로 로딩 Drosophila 신경 터미널

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

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Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

세포는 형광 현미경 Electrofusion와 시각

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 개인 염기 해상도와 단백질 RNA 상호 작용의 Transcriptome 전체 매핑

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

개별 준비 Drosophila</em라이브 이미징을위한> 계란 회의소

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.