우리는 자금에 대한 화학 생화학의 우스터 폴리 테크닉 연구소 부서를 감사드립니다.

1. 세포 라인의 Transfection Transfection 방법 1 (증가 기본 세포 transfection 효율을위한 Lonza의 Nucleofection) <ol><li> 세포는 최근 통과 사전 transfection 로그 위상 성장해야합니다. phophate의 세포를 세척하는 것은 trypsinization하여 생리 (PBS)와 수확 세포를 버퍼. </li><li> 5 분 1,500 XG에서 원심 분리하여 세포를 수집합니다. </li><li> 6 잘 플레이트에 살균 커버 전표를 추가합니다. 커버 풀렸는데이 향상 세포 접착을위한 코팅 수 있습니다. 플레이스 1.5 문화 미디어의 ML (이 경우에는 Dulbecco의 수정 이글 배지 (DMEM) 10 % 태아 소 혈청 (FBS)) 각 우물에와 인큐베이터에있는 미디어를 평형. </li><li> 인산염의 최소 볼륨에서 trypsinization와 resuspend하여 세포를 수확하는 것은 (PBS) 살린 버퍼. </li><li> 세포를 카운트하고 샘플 당 ~ 5x10 5 세포를 제공하기 위해 resuspension의 정확한 금액을 원심 분리기. </li><li> 샘플 당 100 μL 실내 온도 Nucleofector 솔루션에 신중하게 세포를 Resuspend. </li><li> 5 μg 플라스미드 DNA와 세포 현탁액을 100 μL을 결합하고 기포를 포함하지 않도록주의되는 Nucleofector 쿠베트에 샘플을 전송합니다. </li><li> 해당 Nucleofector 프로그램 (이것은 최소한의 사망률과 최적의 transfection 효율을 수립 이전에 테스트를 요구할 수 있습니다)를 선택합니다. </li><li> Nucleofector 쿠베트 홀더에 세포 / DNA 정지를 포함하는 쿠베트를 삽입하고 선택한 프로그램을 시작합니다. </li><li> 완료되면, 쿠베트을 제거하고 (6 - 음 판에서 가져온)로 사전 equilibrated 문화 미디어의 ~ 500 μL를 추가합니다. </li><li> 즉시 잘 당 1.5 ML의 최종 볼륨 6 잘 플레이트에 샘플을 전송합니다. 전지는 컨트롤, 혼합 또는 별도로 인구 중으로 도금되어야합니다. </li></ol> Transfection의 방법 2 (세포 라인에 대한 Qiagen Effectene의 transfection) <ol><li> 첫번째 200 μL EC 버퍼 각 플라스미드 DNA 샘플의 0.8 μg을 추가하여 Qiagen Effectene 시약을 사용하여 transfection의 단지를 준비합니다. </li><li> 각 샘플에 6.4 μL 향상제 시약을 추가, 튜브를 flicking하여 간략하게 혼합하고, 실온에서 2 분 알을 품다. </li><li> 튜브를 flicking하여 각 샘플, 혼합 20 μL Effectene 시약을 추가하고, 실온에서 15 분 동안 품어. </li><li> 이 부화하는 동안 transfection에 대한 관심의 두 세포 라인을 준비합니다. 한번 인산염 최근 passaged 세포를 (&lt;80 % 합류) 세척 식염수 (PBS)가 버퍼. 다시 1.5 ML 신선한 예열 및 equilibrated 성장 매체를 추가하십시오 (이 경우에는 Dulbecco의 수정 이글 배지 (DMEM) 10 % 태아 소 혈청 (FBS)). </li><li> transfection의 단지 15 분 동안 incubated 후에는, 각 샘플 미디어 1.2 ML를 전송합니다. pipetting, 즉시 6 자 판에서 두 우물의 각 단지의 ~ 700 μL를 전송하여 섞는다. dropwise 추가하고 번호판을 소용돌이로 부드럽게하여 섞는다. 세포가 적어도 3 시간 동안 transfect 수 있도록 허용 (세포 유형과 다를 수 있습니다.) </li><li> 새로운 6 - 잘 플레이트에 살균 커버 전표를 추가합니다. 커버 풀렸는데이 향상 세포 접착을위한 코팅 수 있습니다. </li><li> 세포 transfected 후, 각 잘하는 약 100 μL 트립신 솔루션을 추가 간단히 완전히 코트 각각 잘 수있는 접시 agitating, 즉시 트립신을 aspirating하여 PBS로 두 번 세포를 씻어하고 최소한의 trysinization하여 세포를 수확. 세포가 해제되면, 1.5 ML 신선한 미디어를 추가할 수 있습니다. </li><li> 이 시점에서, 세포는 멸균 커버 전표에 혼합 또는 별도의 중 인구 (컨트롤)에 도금되어야합니다. </li><li> 세포는 12 시간 복구할 수 있습니다. </li></ol> 2. 세포 융합 <ol><li> 세포에서 문화 미디어를 제거하고 PBS로 두 번 씻는다. </li><li> 이 시점에서, 그것은 단백질 합성을 억제하기 위해 cycloheximide (100 μg / ML)로 세포를 치료하는 것이 좋습니다. 세포 2 시간 동안 부화 후 PBS로 두 번 세포를 씻어 수 있습니다. </li><li> 상온에서 1백25초에 대한 혈청없는 DMEM에 50% 폴리에틸렌 글리콜 1000 (PEG 1000)의 솔루션을 노출하여 퓨즈 세포. </li><li> PEG 1000 솔루션을 제거하고 신선한 예열 및 equilibrated 미디어이 ML을 추가하는 PBS로 세포를 씻으십시오. </li><li> 24시간 - 세포가 18 품어 수 있습니다. </li></ol> 형광 현미경 <ol><li> 문화 미디어를 제거하고, PBS로 두 번 세포를 씻으십시오. </li><li> 각각 잘 수있는 4 % paraformaldehyde 용액 (PBS)를 1 ML를 추가하여 세포를 수정. 부드럽게 15 분 동안 선동. </li><li> 두 번 PBS에 고정 세포를 씻으십시오. </li><li> 조심스럽게 접시에서 커버 전표를 제거, 여분의 PBS에서 심지. 현미경에 장착 매체 중 하나를 드롭 (90 % 글리세롤, 100mM 트리스 산도 7.5, 2% DABCO (1,4 - diazabicoyclo - 옥탄)이있는 DAPI (4 &#39;, 6 diamidino - 2 - phenylindole)와로드 슬라이드. 반전 </li><li> 슬라이드에서 초과 장착 매체를 제거하고, 인감 공동버전은 매니큐어와 전표. </li><li> 공촛점 또는 epifluorescence 현미경을 통해 세포를 시각화. </li></ol> 3. 대표 결과 그림 2는 기본 섬유아 세포 및 H1299 비 작은 세포 폐 암종 세포를 사용하는 예제 heterokaryon 실험을 보여줍니다. 본 실험에 대한 관심의 단백질 (치킨 빈혈 바이러스 - VP3)는 epifluorescence 현미경에 의해 시각으로 기본 셀 형식에 주로 세포질 현지화 및 변형 세포 유형에 핵 지방화를 표시합니다. 단백질은 H1299 세포의 기본 포피의 섬유아 세포 (PFF) 또는 dsRed에서 GFP 중 하나에 융합 (pE​​GFP - N1 벡터, Clontech) (dsRed - N1 벡터, Clontech)로 표시됩니다. 자기 fusions 관련된 컨트롤 샘플 (상위 2 행)은 정상 상태 로컬 라이제이션 패턴에는 변화와 multinucleated 세포를 표시합니다. 이러한 변경 사항은 별도로 융합 조건이나 syncitia의 형성에 감도로 인해 발생할 수 있습니다. Heterokaryon의 fusions (하단 행)는 GFP - 융합 기본 셀 - 파생 단백질과 변형 세포 물질의 도입에 대응 dsRed - 융합 단백질로 colocalization의 핵 항목을 보여줍니다. 표시 예제는 두 fluoroprotein 신호의 존재에 의해 입증 두 세포, 각 유형 중 하나에서 발생하는 비교적 드문 heterokaryon 융합이다. 현지화 전환 이러한 유형의 탐지뿐만 아니라, nucleocytoplasmic 였죠 활동은 쉽게 두 핵 사이에 하나의 형광단의 존재에 의해 감지가 가능하다. 1시 1분 세포 fusions을 검토하고 번역의 억제에 따라 것이다 때 결정이 유형의 분명하다. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="그림 1" /> 그림 1. 기본 섬유아 세포 및 H1299 비 작은 세포 암종 세포를 사용하여 heterokaryon 방식의 플로우 차트. 관심 유전자 두 형광 단백질 유전자 중 하나의 프레임 융합을 생산하기 위해 복제됩니다. 셀 라인은 다음 transiently 중 구조로 transfected하고 복구하는 허용됩니다. Heterokaryon 형성은 더욱 부화 다음 폴리에틸렌 글리콜 (PEG)과 간단한 치료에 의해 유발됩니다. 마지막으로, 관심의 단백질의 하위 세포 로컬 리 제이션은 형광 현미경의 다양한 형태를 사용하여 관찰합니다. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="그림 2" /> 그림 2. Nucleocytoplasmic 밀매와 치킨 빈혈 바이러스 VP3 단백질의 활동을 였죠은 heterokaryon 융합에 의해 시각. 끝줄 : dsRed - VP3을 표현 자체 융합 H1299 세포의 대표 이미지 중간 행 :.. PEG 융합 후 GFP - VP3을 표현하는 기본 포피의 fibroblast 세포 (PFF) 대표 epifluorescence 이미지 아래 행 : PFF 및 H1299 세포의 Heterokaryon 융합. 노란색은 GFP와 dsRed 신호 colocalization을 나타냅니다. 전지 Leica AF6000E 형광 현미경 및 Leica 이미징 소프트웨어를 사용하여 몇 군데 있었다.

<ol>
	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

heterokaryon 프로토콜은 세포 유형의 특정 현지화 행동뿐만 아니라 nucleocytoplasmic 였죠 활동의 특성화를위한 효율적이고 쉽게 interpretable 방법을 제공 여기를 발표했다. 이 방법은 형광 분석의 감도뿐만 아니라 기능 영상 라이브 세포와 단백질 역학 연구를 수행을 활용합니다. 기술은 쉽게 다양한 세포 유형에 적응하고 생활하고 고정 세포 이미징 모두 의무입니다. 예를 들어, 거꾸로 형광 현미경은 라이브 이미징 및 시간 저속 비디오 캡처에 사용할 수 있습니다. 반대로, 탑재 전지가 정상 상태 localizations 또는 개별 localizations의 상세한 공촛점 이미징의 결정 모두에 대해 epifluorescence 현미경에 사용할 수 있습니다. 또한, 이러한 photobleaching 후 형광 복구 (FRAP) 또는 photobleaching에 형광 손실 (플립)와 같은 기술은 현지화 속도론 1의 결정에 사용할 수 있습니다. 프로토콜의 대체 fluorophores의 결합 콘서트 2 수행하는 형광 공명 에너지 전달 (무서워) 같은 단백질 상호 작용 실험을 허용합니다. 이러한 leptomycin의 B 또는 부정적인 지배와 같은 세포 인신 매매의 다양한 억제제는 GTPase 구성 요소가 특정 가져 오거나 내보내기 경로 5,6,9에 의존을 확립하는 데 사용할 수있는 랜. <ul><li> 활동을 였죠 관련된 특정 실험에서, 관리는 새로운 단백질의 합성에 의한 현지화 효과를 피하기 위해 이동해야합니다. 이러한 경우에는 번역 억제제 또는 제한된 시간 포인트를 사용해야합니다. 그러나, translational 억제로 인해 유물은 가능성이 남아있다. 각 세포 유형 중 하나를 포함하는 셀 fusions은 대부분 였죠 및 현지화 문제의 명확한 해석에 대해 원하는합니다. 휴대폰 번호 및 융합 조건 (타이밍 및 농도)의 최적화는 유리한 1:1 퓨전 이벤트를 홍보해야 할 수도 있습니다. </li><li> 프로토콜의 특정 단계의 최적화를 사용하는 특정 세포 종류에 따라 필요합니다에 유의하는 것이 중요합니다. 이러한 융합 효율, transfection 효율, 그리고 퓨전 후 생존 능력으로 실험 절차 측면 세포 유형 간의 큰 차이가있을 수 있습니다. 특히, 기본 세포 유형은 복합 문화 조건과 낮은 transfection 효율성으로 인해 관리하기 위해 도전하실 수 있습니다. 같은 Lonza Nucleofector 장치로 기술의 사용은 세포의 신속한 처리를 위해뿐만 아니라 급격하게 증가 transfection 효율 허용할 수 있습니다. 또한, electroporation 방법은 다음과 융합의 단계를 중지 세포 쉽게 전송하실 수 있습니다. </li></ul>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3023602</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s modified Eagle&#x2019;s medium (DMEM)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH30243FS</td>
<td>High glucose</td>
</tr>
<tr>
<td>paraformaldehyde</td>
<td>&nbsp;</td>
<td>FisherChemical</td>
<td>O4042-500</td>
<td>&nbsp;</td>
<tr>
<td>4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D9542-10MG</td>
<td>&nbsp;</td>
<tr>
<td>Hyclone Serum (fetal bovine)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH3008803HI</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 6-well plates</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-772-1B</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol 1000</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>528875-25GM</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545-85</td>
<td>&nbsp;</td>
<tr>
<td>DABCO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2522-25G</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector HDF kit</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPD-1001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

heterokaryon technique for analysis of cell type-specific localization

단백질의 상당수는 subcellular 매매 또는 였죠 nucleocytolasmic에 의해 규제됩니다. 이러한 단백질은 핵 수입 / RNA와 단백질의 수출, transcriptional 규제 및 apoptosis를 포함한 세포 기능의 다양한 배열을 표시합니다. 흥미롭게도, 세포 분열, 분화 및 변형 등 주요 휴대폰 reorganizations는 종종 그러한 활동을 할 3,4,8,10를 포함. 이러한 단백질과 해당 규제 메커니즘의 상세한 연구는 이러한 현지화 변경 기묘한 수에 대한 자극하고, 스스로 추적하는 매우 역동적이고 어려울 수있는 움직임으로 도전하실 수 있습니다. 세포 oncogenesis과 관련된 연구는 예를 들어, 이해의 경로와 정상 세포 및 기본 세포 변형 6,7,11,12 다를 단백질 활동의 혜택을 계속합니다. 많은 단백질은 변경 변형 동안이나 변환의 결과로 현지화 효율적으로이 단백질을 특성화 방법과 그들이 마약 타겟팅 oncogenesis 및 오픈 새로운 영역에 대한 이해를 향상시키기 위해 서 참여하는 경로를 보여으로. 여기 단백질 인신 매매의 분석을위한 방법을 제시하고 기본 및 변형 포유 동물 세포 사이의 활동을 였죠. 이 방법은 정상 상태 또는 동적 단백질 localizations를 감지하는 데 사용할 수있는 유연한 프로토콜을 제공하기 위해 형광 현미경과 heterokaryon의 fusions의 생성을 결합한 제품입니다. 그림 1과 같이, 두 개의 세포 종류 transiently 관심의 유전자에 부착된 fluoroprotein의 유전자를 플라스미드 베어링 구성과 transfected 있습니다. 표현 후, 전지는 폴리에틸렌 글리콜을 사용하여 융합하고, 단백질 localizations 그런 다음 다양한 방법을 사용하여 몇 군데있을 수 있습니다. 여기에 제시 프로토콜은 전문 기술이 추가될 수도 있습니다에 대한 근본적인 접근 방식이다.

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

였죠 셀 유형 - 특정 단백질 현지화 및 nucleocytoplasmic의 특성화를위한 유연하고 효율적인 방법을 설명합니다. 이 heterokaryon 접근은 세포 융합 후 이미지 단백질 localizations에 찬란 - 표시 융합 단백질을 사용합니다. 프로토콜은 정상 상태 localizations 또는 라이브 셀 이미징을 기반으로보다 역동적인 결정 의무가 있습니다.

셀 유형 - 구체적인 현지화의 분석을 위해 Heterokaryon 기법

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

Research

JoVE Journal

Biology

16.4K 조회수.  Worcester Polytechnic Institute- WPI. 였죠 셀 유형 - 특정 단백질 현지화 및 nucleocytoplasmic의 특성화를위한 유연하고 효율적인 방법을 설명합니다. 이 heterokaryon 접근은 세포 융합 후 이미지 단백질 localizations에 찬란 - 표시 융합 단백질을 사용합니다. 프로토콜은 정상 상태 localizations 또는 라이브 셀 이미징을 기반으로보다 역동적인 결정 의무가 있습니다.

비디오: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

연구

생물학

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

양적 휘트니스 분석 워크플로우

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

휴대 통신 분석을위한 단일 세포 미세 주입

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Micromanipulation 기법 전체적 역동성의 분석 및 Cytoskeletal 레 귤 레이 터의

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 셀 형식 관련 Epigenome 프로 파일링

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

제 브라 피쉬 렌즈의 평가 핵 현지화 및 조직 무결성

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

마우스 간에서 세포 유형 별 유전자 발현 프로파일링

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

초구조적으로 보존된 세포 내 막 단백질 국소화의 전자 현미경 분석을 위한 CryoAPEX 방법

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...