이 문서에 포함된 그림에 대한 세포 문화를 제공하는 닥터 테리 Schochet하는 셀 문화를 도금하고 관리하기위한 케빈 미야에 감사합니다. 또한, 아르나 절차에 입력 케빈 미야 박사 피터 버클리, 그리고 Tiina Pertiz에 감사드립니다. 이 작품에 대한 자금이 노화에있는 국립 연구소, 정신 건강 및 펜실베니아의 연방에서 인적 자원 사실 찾기 자금의 국립 연구소에서했다.

<ol>
	<li>Davis, J. E., Eberwine, J. H., Hinkle, D. A., Marciano, P. G., Meaney, D. F., McIntonsh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+regarding+single-cell+gene+expression+profiling+for+brain+injury.">Methodological considerations regarding single-cell gene expression profiling for brain injury.</a> Neurochemical Research. 29, 1113-1121 (2004).</li><li>Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+molecular+biology.">Single-cell molecular biology.</a> Nature Neuroscience. 4, 1155-1156 (2001).</li><li>Kamme, F., Salunga, R., Yu, J., Tran, D., Zhu, J., Luo, J., Bittner, A., Guo, H., Miller, N., Wan, J., Erlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microarray+analysis+in+hippocampus+CA1:+Demonstration+and+Validation+of+Cellular+Heterogeneity.">Single-cell microarray analysis in hippocampus CA1: Demonstration and Validation of Cellular Heterogeneity.</a> The Journal of Neuroscience. 23, 3607-3607 (2003).</li><li>Hinkle, D., Glanzer, J., Sarabi, A., Pajunen, T., Zielinski, J., Belt, B., Miyashiro, K., McIntosh, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neurons+as+experimental+systems+in+molecular+biology.">Single neurons as experimental systems in molecular biology.</a> Progress in Neurobiology. 72, 129-142 (2004).</li><li>Ginsberg, S. D., Elarova, I., Ruben, M., Tan, F., Counts, S. E., Eberwine, J. H., Trojanowski, J. Q., Hemby, S. E., Mufson, E. J., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+gene+expression+analysis:+Implications+for+Neurodegenerative+and+Neuropsychiatric+Disorders.">Single-cell gene expression analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders.</a> Neeurochemical Research. 29, 1053-1064 (2004).</li><li>Eberwine, J., Spencer, K., Miyashirto, K., Mackler, S., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+synthesis+in+situ:+methods+and+applications.">Complementary DNA synthesis in situ: methods and applications.</a> Methods Enxymol. 216, 80-100 (1992).</li><li>Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., Coleman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+in+single+live+neurons.">Analysis of gene expression in single live neurons.</a> Proc. Natl. Acad. Sci. USA. 89, 3010-3014 (1992).</li><li>Kelz, M. B., Dent, G. W., Therianos, S., Marciano, P. G., McIntosh, T. K., Coleman, P. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+antisense+RNA+amplification+and+microarray+analysis+as+a+tool+for+studying+neurological+degeneration+and+restoration.">Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.</a> Sci. Aging Knowledge Eviron. 1, re1-re1 (2002).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126, 397-425 (1977).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MinElute Handbook For MinElute Reaction Cleanup Kit. , 28-29 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Megascript kit. , 7-8 .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MEGAclear kit. , 4-5 .</li><li>Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+expression+analysis+of+tissue+sections+and+single+cells.">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li><li>Miyashiro, K., Dichter, M., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis:+Implications+for+neuronal+functioning.">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li><li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+transfer+produces+a+predictable+cellular+phenotype.">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li><li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, 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=Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis:+Application+of+an+excel-based+qPCR+preparation+software+(PREXCEL-Q).">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li><li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li><li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy.">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li></ol>

박사 Eberwine 박사 Eberwine가 공유 소유자가있는 LBS 기술이 허가되었습니다 아르나의 특허 발명가이다.

노트 및 문제 해결 <ul><li> 당신은 전후 대상 셀을 격리 것을 보여주는 이미지와 나머지 penumbra가 온전히 남아 있다고 걸릴 수 있습니다. </li><li> 너무 많은 솔루션이 수확 그대로 피펫을 입력하는 경우, 3 Luer - 잠금 피팅 방법과 튜브에 긍정적인 압력을 유지 주사기에 대한 플런저를 사용할 수 있습니다. 원하는 볼륨 처리의 유형에 따라 달라질 수 있지만 최대 5 μl은 대부분의 어플 리케이션을위한 괜찮을 것입니다. </li></ul> 분자 생물 학적 기법에 대한 일반적인 도움말 <ul><li> 와동 모든 재고 튜브와 반응에 추가하기 전에 그들을 스핀. 절대 와동 효소. </li><li> 철저하게 버퍼가 적절한 농도에 있는지 확인하기 위해 효소를 추가하기 전에 반응을 섞는다. </li><li> 가능하면 stratacooler를 사용하여 -20 ° C에서 효소 주식 보관하십시오. 그렇지 않으면, 바로 사용하기 전에 제거 얼음을 계속하고, -20 ° C. 즉시 반환 </li><li> 항상 pipetting 오류를 줄이기 위해 하나 이상의 반응을 준비할 때 주인이 혼합합니다. 샘플의 개수에 따라 필요한 볼륨을 계산하고 10-20%를 추가합니다. </li><li> 스토어 RNA -80 ° C에서 저능아 저하합니다. RNA는 산성 조건에서 발생하는 RNA의 저능아 apurination에 버퍼에 저장되어있는 가장 안정적인 것입니다. </li></ul> 아르나 절차의 일반 개요 그림 4A는 아르나 절차의 첫 번째 라운드를 보여줍니다. 첫 번째 스트랜드 반응에서, T7 -​​ oligo (DT) 프라이머의 폴리 - T 부분은 polyA 꼬리에 바인딩하여 mRNA 종 (길고 흰 사각형)을 선택합니다. 일부 microRNAs는 polyadenylated하고이 프로 시저에 의해 캡처됩니다. 더 중요하지만, 세포에서 가장 풍부한 RNAS, ribosomal RNAS가되지 않습니다. 이 oligo는 템플릿으로 mRNA를 사용하여 cDNA의 보완 스트랜드 (긴 회색 사각형)를 합성하기 위해 역방향 Transcriptase위한 입문서 역할을합니다. T7 - oligo (DT) 프라이머의 T7 부분은 시작 mRNA에 순서 안티 센스와 프레임의 T7 RNA 효소 프로 모터를 포함합니다. 이것은 나중에 시험 관내 전사 반응에 사용됩니다. 다음, 이전 단계에서 만든 mRNA / DNA 하이브리드의 mRNA는 보온 스트랜드의 DNA 합성에서 만든 오카자키 조각과 유사한 RNA &quot;primers&quot;을 (작은 흰색 사각형) 작성 Rnase H에 의해 부분적으로 분해됩니다. 의 DNA 중합 효소는 한 템플릿으로 mRNA를 보완 DNA를 사용하여 프라임 DNA 합성에 RNA 조각을 사용합니다. 그것이 &#39;3&#39;다음 RNA 조각, 그 5에 도달하면 nuclease 활동 ribonucleotides를 제​​거하고 deoxyribonucleotides으로 그들을 대체합니다. DNA ligase는 선도 가닥의 교체가 완료되지 않은 긴 ligate에 추가됩니다. RNA 조각이 DNA를 중합 효소 내가 만드는 초기 primers 역할을 어디 T4의 DNA 중합 효소는 지역에 채우기 추가됩니다 날이 종단을 두 번 누른 다음 IVT 반응을 수행하기 전에 정화입니다 cDNA 좌초. IVT 반응에 T7 RNA 효소는 템플릿으로 감지 가닥을 사용하여 두 cDNA 합성 좌초와 안티 센스 RNA 분자 (검고 긴 직사각형)에 반영된 T7 프로 모터에 바인딩합니다. 이것은 안티 센스 RNA 분자의 수천이 각각 두 번 좌초 cDNA 분자 (그림 4A)에서 생산되는 증폭 단계 역할을합니다. 그림 4B에 묘사된 두 번째 라운드는, 시작 RNA 이후 첫 라운드와는 약간 다른 역 전사 반응을 시작하는 안티 센스 (고체 검정 직사각형)이고 T7 -​​ oligo의 대상이되었다 polyadenylated 꼬리 부족 첫 라운드에서 (DT) 입문서. 따라서,이 반응은 임의 primers (작은 회색 사각형)과 이후 변성 RNA와 함께 준비하는 것입니다. 두 번째 스트랜드 반응은 다음 이전 반대 전사 반응에서 만든 감각의 RNA의 3 &#39;끝에 폴리 - A 시퀀스에 바인딩 T7 -​​ oligo (DT) 입문서로 준비하는 것입니다. 다른 IVT 반응은 첫 라운드에서 같은 방식으로 수행됩니다. 이 두 번째 라운드는 일반적으로 하나의 세포에서 증폭의 3 라운드를 달성하기위한 최소한 한 번 반복됩니다. 애플 리케이션 우리는이 문서에서 제시해야하는 기술은 애플 리케이션의 많은로 번역하실 수 있습니다. 단세포 격리 프로토콜은 급성 조각에 사용하기 위해 수정할 수 있습니다. 14 기술적으로 많은 도전하지만, 동일한 원칙이 다른 준비에 적용됩니다. 피펫의 크기가 약간 조정 경우 또한, 세포의 생리의 기록은 수확 생리 출력 뒤에 분자 메커니즘을 잘 제어 조사를 위해 허용하기 전에 할 수 있습니다. 또 약간의 수정이 셀 소마에서 프로세스를 분리하는 것입니다.이 응용 프로그램 15 하나의 피펫과 함께 세포의 시체를 수집하고 신선한 피펫로 돌아가서 100-300 식별 dendrit를 수집에스 또는 수집 관 당 axons. 16 일단 세포는 mRNA의 abundances와 작곡의 비교는 동일한 세포 집단 내에서조차 다른과 사이에 만들 수 있으며, 수확되었습니다. 세 번째 라운드 아르나로 biotinylated - UTP를 통합하면 이러한 상대적인 mRNA의 abundances을 결정하기 위해 microarray 분석을 위해 수 있습니다. 원래 mRNA 인구의 구성 또한 차세대 시퀀싱을 사용하여 아르나 절차 후 결정하실 수 있습니다. 증폭 아르나도 하나의 셀 유형에서 mRNAs의 전체 집합이 이전의에 후자의 셀 타입의 표현형의 전환을 유도하기 위해 다른 세포 유형으로 transfected하는 세포 표현형 변환 연구를 확인하는 데 사용할 수 있습니다 절차는 실험실에서 개발하여 TIPeR으로 알려져 있습니다. 17 이러한 연구는 질병 상태 및 세포 phenotypes 및 연구를 공부에 특히 유용합니다 연구실에서 현재 진행하고 있습니다. RT - PCR 또는 qPCR은 셀 - 특정 유전자의 표현을 확인하는 증폭 자료에 수행할 수 있습니다. 또한, transfection 또는 전달의 효율성의 평가는 단일 세포 수준에서 만들 수 있습니다. 장점 및 제한 으로 추상적으로 명시된 바와 같이, 분석을 위해 단일 ​​세포의 분리는 이기종 세포 인구의 분석과 본 평균 효과를 제거합니다. 이러한 평균 효과는 풍부한 기록을 통해 - 대표 밖 평균 많은 낮은 풍부한 성적 증명서의 확인을 방지하여 단일 세포 내에서 mRNA의 abundances를 허위. 유동세포계측법은 개별 세포를 정렬하는 데 사용하지만,이 방법은 세포 특정 마커와 고가의 장비에 대한 지식을 필요로하실 수 있습니다. 18 레이저 캡처 microdissection도 자외선 또는 적외선 레이저 캡처 microdissection 시스템과 심지어 단일 셀 및 subcellular 캡처를 허용하지만, 관심의 세포를 필요로 매우 얇은 부분의 표면에 위치합니다. 19 유동세포계측법 마찬가지로, 레이저 캡처 microdissection은 값비싼 장비가 필요합니다. 위에서 설명한 전극을 기반으로 수집 기술의 주요 장점 중 하나는 귀중한 electrophysiological 데이터가 동일한 세포에서 수행하는 기능과 transcriptome 분석을위한 허용, 수확하기 전에 관심있는 세포에서 얻을 수있다. 우리의 기술 20 단점입니다 그것은 micromanipulators를 사용하여 경험을 필요로 않습니다. micromanipulators 익숙한 수사관이 기술은 매우 직관적인 발견할 것이다, 그러나, 이러한 경험을 가진 개인은 필요한 좋은 움직임과 함께 편안한 될해야합니다. 모든 증폭 기술 고유의 크기와 염기 조성에 따라 특정 성적의 특혜 증폭입니다. 4,6,7 중합 효소의 연쇄 반응 (PCR)과 같은 리버스 전사 중합 효소의 연쇄 반응 (RT - PCR) 및 cDNA의 급속한 증폭과 같은 기반 기술 성적 지수 증폭에 종료 (RACE) 결과, 선형 증폭의 반면 아르나의 증폭 절차 결과. 따라서 아르나 절차의 주요 장점 중 하나가 더 나은 유일한 선형 기하 급수적으로 확장 반대로 증폭 과정에서 발생하는 오류나 편견을 확장하여 mRNA의 성적의 상대적 abundances를 보존하는 기능에있다 오류와 편견했다. microarray 분석과 동봉된 아르나 절차는 치료 또는 치료 유사하거나 다른 형태의 단일 셀 사이의 mRNA의 abundances의 비교를위한 수 있습니다. 또한 증폭 자료는 차세대 시퀀싱을위한 제출하실 수 있습니다. 5,8는 그러나, 프로 시저에 대한 무작위 primers를 사용하는 반면에, oligo - DT 준비하는 절차는의 편견 증폭 위에서 설명한 이후 같은 분석에 관심 기울여야 3 &#39;은 mRNA 각각의 원형과 그 이후 증폭된 소재의 약간의 단축에 일반적으로 결과의 끝납니다. 치료는 몇 가지 잘못된 부재 통화가 약간 짧아 증폭 소재에서 발생할 수있는 표준 방법으로 microarray 결과를 분석에 주의해야한다. 또한, 시퀀싱하면서 결과가 실제로 전체 5 제공할 것입니다 &#39;원래의 mRNA 대부분의 순서를 5&#39;일부 mRNA의 시퀀스는 놓칠 수 있습니다. 이러한 이유로, amplifications의 반올림의 수를 제한해야합니다.

A correction was made to <a href="http://http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

<!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>Spiegelgas coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological</td>
<td>63-3029</td>
<td>&nbsp;</td>
<tr>
<td>Nunc 35x10 mm culture dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-90</td>
<td>&nbsp;</td>
<tr>
<td>Water for cell culture</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-724Q</td>
<td>For making solutions used for cell culture and rinsing coverslips</td>
</tr>
<tr>
<td>Poly-D-Lysine MW70-150K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6407</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, ultrapure</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354239</td>
<td>&nbsp;</td>
<tr>
<td>Boric acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0252</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and glutamax</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41090-101</td>
<td>For plating cells</td>
</tr>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8769</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Cytosine beta-D-arabinofuranoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1768</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095-098</td>
<td>For growing cells</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>B27 serum-free supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td>
<td>&nbsp;</td>
<td>Kimble Chase</td>
<td>34500 99</td>
<td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td>
</tr>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175</td>
<td>Any solution will work as long as the components won&#x2019;t interfere with any future processing (i.e. no Ca2+ or Mg2+)</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309628</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>Gauge depends on the diameter of the pipettes</td>
</tr>
<tr>
<td>dNTP mix</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>28-4065-51</td>
<td>&nbsp;</td>
<tr>
<td>dt-T7 oligo</td>
<td>&nbsp;</td>
<td>Custom</td>
<td>Midland Certified</td>
<td>&nbsp;</td>
<tr>
<td>Second strand buffer (5x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Y01129</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Supplied with second strand buffer</td>
</tr>
<tr>
<td>E.coli DNA Ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100002324</td>
<td>&nbsp;</td>
<tr>
<td>DNA polymerase I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004926</td>
<td>&nbsp;</td>
<tr>
<td>Rnase H</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18021-071</td>
<td>&nbsp;</td>
<tr>
<td>Megascript T7 kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1334</td>
<td>&nbsp;</td>
<tr>
<td>Random primers</td>
<td>&nbsp;</td>
<td>BMB</td>
<td>11034731001</td>
<td>&nbsp;</td>
<tr>
<td>Superscript III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>56575</td>
<td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td>
</tr>
<tr>
<td>MEGAclear Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>1908</td>
<td>&nbsp;</td>
<tr>
<td>MinElute Reaction Cleanup Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28206</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004994</td>
<td>&nbsp;</td>
<tr>
<td>Rnasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N251B</td>
<td>&nbsp;</td>
<tr>
<td>Illumina TotalPrep RNA Amplification Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AMIL1791</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-87</td>
<td>Sutter has many other models, many are discussed in the cookbook</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Many other micromanipulators will work such as the newer Eppendorf models</td>
</tr>
<tr>
<td>Pipette Holder</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>MP-S15A</td>
<td>Will vary with micromanipulator and pipette O.D.</td>
</tr>
<tr>
<td>Bioanalyzer RNA 6000 Nano Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1511</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transcriptome analysis of single cells

대부분의 유전자 발현 분석 기술 문화 조직 homogenates이나 세포에서 세포의 이질적인 인구의 격리 물질에 의존하고 있습니다. 1,2,3 두뇌의 경우, 같은 해마로 영역 각각 다른 세포 유형의 복잡한 배열을 포함 서로 다른 mRNA 프로필. 단일 세포를 수확하는 능력은 세포 인구 사이 내의 분자의 차이점에 대한 심층 조사에 더 있습니다. 우리는 더 이상 처리를위한 세포를 수확하는 간단하고 빠른 방법을 설명합니다. 자주 전기 생리학에서 사용 Pipettes는 (열망 사용) 관심의 세포를 분리하기 위해 활용하고 편리하게 분자 생물학 기술의 번호와 추가 처리를 위해 Eppendorf 튜브에 그것을 입금하고 있습니다. 우리 프로토콜 세포 배양이나 급성 조각에서 개별 세포에서 dendrites의 수확 수정할 수 있습니다. 우리는 또한 단일 셀 isolations의 주요 다운 스트림 응용 프로그램으로 아르나의 증폭 방법을 설명합니다. 이 방법은 같은 리버스 녹음하거나 실시간 중합 효소의 연쇄 반응 (PCR) 등의 유전자 발현 분석 기술의 대안으로 우리 연구실에 의해 이전에 개발되었습니다. 4,5,6,7,8이 기술의 선형 증폭을 위해 제공 polyadenylated RNA는 재료만을 femtograms로 시작하는 안티 센스 RNA 및 마이크로 그램의 금액에 결과. 선형 증폭 재료는 절연 세포의 transcriptome의 구성 요소의 상대적인 풍요의 PCR 증폭 지수보다 정확한 견적을 제공합니다. 기본 절차는 증폭의 두 라운드로 구성되어 있습니다. 간단히, T7 RNA 효소 프로 모터 사이트는 mRNA의 기록에서 만든 두 번 좌초 cDNA에 통합됩니다. 시험 관내 전사의 하루 (IVT) 반응은 다음 T7 RNA 효소는 이중 좌초 cDNA로부터 많은 안티 센스 성적표를 생산하는 수행됩니다. 두 번째 라운드는이 과정을 반복하지만 시작부터 자료 몇 가지 기술적인 차이와 안티 센스 RNA입니다. 그것은 증폭의 3 라운드의 결과로, 두 번째 라운드를 반복하는 기준입니다. 종종, 시험 관내 전사 반응에서 세 번째 라운드는 생산 안티 센스 RNA가 hybridized와 microarray를 감지 수 있도록 biotinylated 뉴클레오 사이드의 triphosphates를 사용하여 수행됩니다. 7,8

Watch this Scientific Journal Video about Transcriptome Analysis of Single Cells at JoVE.com

Transcriptome Analysis of Single Cells

이 문서에서 우리는 쥐를 기본의 연결을 문화와 아르나의 증폭을 사용하여 후속 transcriptome 분석에서 단일 세포의 수확을위한 간단한 방법을 설명합니다. 이러한 접근 방식은 모든 세포 유형에 generalizable있다.

단일 셀 Transcriptome 분석

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Research

JoVE Journal

Neuroscience

29.6K 조회수.  University of Pennsylvania. 이 문서에서 우리는 쥐를 기본의 연결을 문화와 아르나의 증폭을 사용하여 후속 transcriptome 분석에서 단일 세포의 수확을위한 간단한 방법을 설명합니다. 이러한 접근 방식은 모든 세포 유형에 generalizable있다.

비디오: Transcriptome Analysis of Single Cells

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

연구

신경과학

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

외상성 뇌 손상 후 뉴런 또는 유전자 발현 분석을위한 단일 뉴런의 강화 인구의 레이저 캡처 Microdissection

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

원자 힘 현미경과 뇌 Polysom​​es에서 피어링

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

망막 신경절 세포의 정의 된 서브 세트의 단일 세포 RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

단일 좌 골 신경 및 Cytometry 사용 하 여 단일 마우스에서 지 루트 신경 절에 면역 세포의 분석

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

대규모 병렬 단일 핵 RNA 시퀀싱에 대 한 성인 척수 핵의 절연

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

미세 주입을 사용하여 인간 헬라 세포에서 스플리세오소말 snRNA 국소화 분석

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.