Name: a practical guide to phylogenetics for nonexperts
Uploaded: 2014-02-05T17:00:00
Description: Watch this Scientific Journal Video about A Practical Guide to Phylogenetics for Nonexperts at JoVE.com

우리는 원고에 대한 의견에 대한 오할로 랜 실험실의 구성원을 감사드립니다. 우리는 D. 오할로 랜에 자금 조달을위한 생명 과학의 조지 워싱턴 대학학과 및 예술과 과학의 콜롬비아 대학을 감사합니다.

1. 기본 지역 정렬 검색 도구 (BLAST) : 온라인 인터페이스 <ol><li> 생명 공학 정보를위한 국립 센터 (NCBI)의 BLAST 1 웹 서버를 방문하려면이 링크를 클릭하십시오. - <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (그림 1). </li><li> 쿼리 상자에 입력 FASTA 포맷의 텍스트 시퀀스 (예를 들어 그림 2 참조). </li><li> 검색에 사용하고 &quot;BLAST&quot;을 클?...

<ol>
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Stat. 6 (2), 461-464 (1978).</li><li>Guindon, S., Gascuel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+fast,+and+accurate+algorithm+to+estimate+large+phylogenies+by+maximum+likelihood.">A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.</a> Syst. Biol. 52 (5), 696-704 (2003).</li><li>Huelsenbeck, J. P., Ronquist, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRBAYES:+Bayesian+inference+of+phylogenetic+trees.">MRBAYES: Bayesian inference of phylogenetic trees.</a> Bioinformatics. 17 (8), 754-755 (2001).</li><li>Thompson, J. D., Higgins, D. G., Gibson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLUSTAL+W:+Improving+the+sensitivity+of+progressive+multiple+sequence+alignment+through+sequence+weighting,+position-specific+gap+penalties+and+weight+matrix+choice.">CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.</a> Nucleic Acids Res. 22 (22), 4673-4680 (1994).</li><li>Lassmann, T., Sonnhammer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kalign--an+accurate+and+fast+multiple+sequence+alignment+algorithm.">Kalign--an accurate and fast multiple sequence alignment algorithm.</a> BMC Bioinformatics. 6, 298 (2005).</li><li>Katoh, K., Kuma, K., Toh, H., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT+version+5:+Improvement+in+accuracy+of+multiple+sequence+alignment.">MAFFT version 5: Improvement in accuracy of multiple sequence alignment.</a> Nucleic Acids Res. 33 (2), 511-518 (2005).</li><li>Katoh, K., Misawa, K., Kuma, K., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT:+A+novel+method+for+rapid+multiple+sequence+alignment+based+on+fast+fourier+transform.">MAFFT: A novel method for rapid multiple sequence alignment based on fast fourier transform.</a> Nucleic Acids Res. 30 (14), 3059-3066 (2002).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+Multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: Multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Notredame, C., Higgins, D. G., Heringa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-coffee:+A+novel+method+for+fast+and+accurate+multiple+sequence+alignment.">T-coffee: A novel method for fast and accurate multiple sequence alignment.</a> J. Mol. Biol. 302 (1), 205-217 (2000).</li><li>Do, C. B., Mahabhashyam, M. S., Brudno, M., Batzoglou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProbCons:+Probabilistic+consistency-based+multiple+sequence+alignment.">ProbCons: Probabilistic consistency-based multiple sequence alignment.</a> Genome Res. 15 (2), 330-340 (2005).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProtTest+3:+Fast+selection+of+best-fit+models+of+protein+evolution.">ProtTest 3: Fast selection of best-fit models of protein evolution.</a> Bioinformatics. 27 (8), 1164-1165 (2011).</li><li>Page, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeView:+An+application+to+display+phylogenetic+trees+on+personal+computers.">TreeView: An application to display phylogenetic trees on personal computers.</a> Comput. Appl. Biosci. 12 (4), 357-358 (1996).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=jModelTest+2:+More+models,+new+heuristics+and+parallel+computing.">jModelTest 2: More models, new heuristics and parallel computing.</a> Nat. Methods. 9 (8), 772 (2012).</li><li>Chevenet, F., Brun, C., Banuls, A. L., Jacq, B., Christen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeDyn:+Towards+dynamic+graphics+and+annotations+for+analyses+of+trees.">TreeDyn: Towards dynamic graphics and annotations for analyses of trees.</a> BMC Bioinformatics. 7, 439 (2006).</li></ol>

이 기사에 대한 우리의 희망은 계통 발생에 새로운 연구자 또는 학생들을 안내하는 출발점이 될 것입니다. 게놈 시퀀싱 프로젝트는 지난 몇 년 동안 저렴하게하고 결과적으로이 기술에 대한 사용자의 수요가 증가하고 있으며, 현재 많은 시퀀스 데이터 세트의 생산은 소규모 실험실에서 일반적이다. 이 데이터 세트는 종종 자신의 기능을 이해하기 시작하는 계통 발생 학적 프레임 워크를 필요로?...

두 개 (또는 그 이상)의 종 진화 방법을 이해하기 위해서는, 각각의 샘플로부터 서열 또는 형태 학적 데이터를 구하는 것이 우선 필요하다, 이러한 데이터는 우리가 공간을 통해 진화 관계를 측정하는 데 사용할 수있는 수량을 나타낸다. (예를 들어, 마일, 인치, 마이크론) 사용 가능한 더 많은 데이터를보다 정확한 측정으로 간주됩니다 데, 직선 거리를 측정 할 때 그냥 좋아. 에르고는 연구자가 진화 거리를 추론 할 수있는 정확도는 크게 관계를 측정하는 것이 가능한 정보 데이터의 양에 의해 영향을 받는다. 다른 샘플은 다른 속도 및 다른 메커니즘에 의해 발전하기 때문에 또한, 우리는이 분류군 사이의 관계를 측정하기 위해 사용하는 방법은 직접적 진화 측정의 정확도에 영향을 미친다. 따라서 진화론 적 관계는 직접 관측되지 않고 대신에 순서 나 형태 학적 데이터, 진화를 추론의 문제로 추정되기 때문에관계는 통계 중 하나가됩니다. 계통 발생 최적 분류군 간의 진화의 역사를 재구성하기 위해 진화의 패턴을 통계 모델을 적용과 관련된 생물학의 지점입니다. 분류군 간의 재건은 분류군의 계통이라고합니다. 우리가 여기 시퀀스의 집합에서 계통 발생을 추론하기위한 단계 파이프 라인에 의해 단계를 설명하는 분자 생물 학자와 진화 생물 학자 사이의 전문 지식의 격차를 해소하는 데 도움이. 첫째, 우리는 세부 사항 웹 기반 인터페이스를 통해 또한 지역 실행 파일을 사용하여 기본 지역 정렬 검색 도구 (BLAST 1) 알고리즘을 사용하여 데이터베이스 심문하는 단계;이 종종 알 수없는 유사한 시퀀스의 목록을 얻는 첫 번째 단계입니다 쿼리, 일부 연구자는 또한 Phylota (http://www.phylota.net/)와 같은 웹 인터페이스를 통해 하나의 그룹에 대한 데이터를 수집에 관심이있을 수도있다. BLAST는 C에 대한 알고리즘질의 시퀀스를 닮은 &quot;히트&quot;에 대한 검색 시퀀스를 데이터베이스에 대해 일차 아미노산 또는 뉴클레오티드 서열 데이터를 omparing. BLAST 프로그램은 스티븐 Altschul 등에 의해 설계되었습니다. 국립 보건원 (NIH)에서 1. BLAST 서버는 다른 프로그램들로 구성되어 있으며, 여기에 가장 일반적인 BLAST 프로그램의 일부 목록입니다 : ⅰ) 뉴클레오티드 염기 BLAST (BLASTN) :이 프로그램은 DNA 서열의 입력을 요구하고 DNA 데이터베이스로부터 가장 유사한 DNA 서열을 반환하는 사용자를 지정하는 특정 유기체 (예를 들면). ⅱ) 단백질 - 단백질 BLAST (BLASTP) : 여기서 사용자는 단백질 서열을 입력하고, 프로그램은 사용자가 지정하는 단백질 데이터베이스에서 가장 유사한 단백질 서열을 반환한다. III) 위치 별 반복 BLAST (PSI-BLAST) (blastpgp) : 사용자 입력은 PROTE입니다밀접한 관련이 단백질의 집합을 반환 순서,이 데이터 집합의 보존 프로파일이 생성됩니다. 다음 새로운 쿼리 단백질 데이터베이스를 심문하는 데 사용되는 이들 보존 된 &quot;모티프&quot;를 사용하여 생성되고 이는 보존 된 &quot;모티프&quot;의 새로운 세트를 추출하고 단백질 데이터베이스까지 심문하는 데 사용되는 단백질의 큰 그룹을 반환한다 단백질의 더 큰 세트 재곡 것으로 다른 프로파일이 생성되어 처리를 반복한다. 각 단계에서 검색어에 관련된 단백질을 포함함으로써이 프로그램은 사용자가 더 발산 아르 시퀀스를 식별 할 수있다. ⅳ) 뉴클레오티드 6 - 프레임 번역 단백질 (BLASTX) : 여기서 사용자는 여섯 프레임 개념적 번역 제품 (즉,로 변환된다 뉴클레오티드 서열 입력을 제공하는 두 가닥) 단백질 서열 데이터베이스에 대해.. V) 6 프레임 번역 염기를 염기6 - 프레임 변환 (tblastx) :이 프로그램은 DNA 염기 서열의 입력을 받아,이 염기 서열 데이터베이스의 여섯 프레임 변환에 대하여 비교 여섯 프레임 개념적 번역 제품에 입력을 변환한다. ⅵ) 단백질 - 뉴클레오티드 6 프레임 변환 (tblastn) :이 프로그램은 뉴클레오티드 서열 데이타베이스의 여섯 판독 범위에 대하여 비교하는 단백질 시퀀스 입력을 사용한다. 다음으로, 우리는 일련의 데이터 집합의 여러 순서 정렬 (MSA)를 생성하기 위해 일반적으로 사용되는 프로그램을 설명하고 이것은 일련의 데이터 집합에 대한 진화에 가장 적합한 모델을 결정하는 프로그램에 대한 사용자 설명서를 따른다. 계통 발생 학적 재구성은 통계 문제이며,이 때문에, 계통 발생 방법은 통계적인 프레임 워크를 통합 할 필요가있다. 이 통계 프레임 워크는 데이터 집합 내에서 일련의 변화를 통합하는 진화 모델이됩니다. 이 진화 개월델은 뉴클레오티드 또는 아미노산 치환의 과정에 대한 가정의 집합으로 구성되며, 특정 데이터 세트에 대한 최적의 모델은 통계적인 테스트를 통해 선택할 수 있습니다. 다른 모델의 데이터에 적합 가능한 것들의 집합 내에서 가장 적합한 모델을 선택하는 우도 비 테스트 (LRTs) 또는 정보 기준을 통해 비교할 수 있습니다. 두 공통 정보 기준은 아카 이케 정보 기준 (AIC) (2) 및 베이지안 정보 기준 (BIC) 3이다. 최적 얼라인먼트가 생성되면, 정렬 된 데이터로부터 계통을 생성하는 다양한 방법이있다. 진화 적 관계를 추론하는 수많은 방법이 있습니다, 광범위하게, 그들은 두 가지 범주로 나눌 수 있습니다 : 거리 기반의 방법과 순서 기반의 방법. 거리 기반 방법은 시퀀스의 페어의 거리를 계산하고 나무를 얻기 위해이 거리를 사용합니다. 시퀀스 기반의 방법은 직접 서열 정렬을 사용하여, 통상 t 검색최적 성 기준을 사용하여 REE 공간. 우리는 계통 발생 학적 관계를 재구성 두 시퀀스 기반의 방법을 간략하게 설명이 최대 우도 프레임 워크를 구현 PhyML 4, 그리고 베이지안 마르코프 체인 몬테카를로 유추를 사용 MrBayes 5. 가능성과 베이지안 방법은 계통 발생 학적 재구성을위한 통계적인 프레임 워크를 제공합니다. 일반적으로 사용되는 트리 구축 도구에 사용자 정보를 제공함으로써, 우리는 계통 발생 학적 관계를 추론하는 데 필요한 필요한 데이터에 독자를 소개합니다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">BLAST webpage&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLAST executables&#160;</td>
			<td></td>
			<td></td>
			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/">ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Preformatted BLAST databases</td>
			<td></td>
			<td></td>
			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/db/">ftp://ftp.ncbi.nlm.nih.gov/blast/db/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustal</td>
			<td></td>
			<td></td>
			<td><a href="http://www.clustal.org/">http://www.clustal.org/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Kalign</td>
			<td></td>
			<td></td>
			<td><a href="http://msa.sbc.su.se/cgi-bin/msa.cgi">http://msa.sbc.su.se/cgi-bin/msa.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAFFT</td>
			<td></td>
			<td></td>
			<td><a href="http://mafft.cbrc.jp/alignment/software/">http://mafft.cbrc.jp/alignment/software/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;">MUSCLE</td>
			<td></td>
			<td></td>
			<td><a href="http://www.drive5.com/muscle/">http://www.drive5.com/muscle/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-Coffee</td>
			<td></td>
			<td></td>
			<td><a href="http://www.tcoffee.org/Projects/tcoffee/">http://www.tcoffee.org/Projects/tcoffee/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROBCONS</td>
			<td></td>
			<td></td>
			<td><a href="http://toolkit.tuebingen.mpg.de/probcons">http://toolkit.tuebingen.mpg.de/probcons&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Se-Al&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://tree.bio.ed.ac.uk/software/seal/">http://tree.bio.ed.ac.uk/software/seal/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSEdit&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.bsedit.org/">http://www.bsedit.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JalView</td>
			<td></td>
			<td></td>
			<td><a href="http://www.jalview.org/">http://www.jalview.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeaView</td>
			<td></td>
			<td></td>
			<td><a href="http://pbil.univ-lyon1.fr/software/seaview.html">http://pbil.univ-lyon1.fr/software/seaview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProtTest&#160;</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/prottest3/">https://code.google.com/p/prottest3/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Java Runtime&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.java.com/en/download/chrome.jsp">http://www.java.com/en/download/chrome.jsp</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Readseq</td>
			<td></td>
			<td></td>
			<td><a href="http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi">http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">jModelTest</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/jmodeltest2/">https://code.google.com/p/jmodeltest2/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PhyML</td>
			<td></td>
			<td></td>
			<td><a href="https://code.google.com/p/phyml/">https://code.google.com/p/phyml/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MrBayes</td>
			<td></td>
			<td></td>
			<td><a href="http://mrbayes.sourceforge.net/download.php">http://mrbayes.sourceforge.net/download.php</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeView</td>
			<td></td>
			<td></td>
			<td><a href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html">http://taxonomy.zoology.gla.ac.uk/rod/treeview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeDyn</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="http://www.treedyn.org/">http://www.treedyn.org/</a></td>
		</tr>
	</tbody>
</table>

a practical guide to phylogenetics for nonexperts

많은 연구자들은 믿을 수 없을만큼 다양한 초점을 통해, 자신의 연구 질문 (들) 계통 발생학을 적용하고있다. 그러나 많은 연구자들은이 주제에 새로운 그리고 그것은 고유의 문제를 제시한다. 여기에서 우리는 비전문가에 대한 계통 발생에 대한 실질적인 소개를 컴파일합니다. 우리는 단계별 방식으로, 유전자 서열 데이터 세트에서 안정적인 계통을 발생 파이프 라인 개요. 우리는 온라인 인터페이스뿐만 아니라 지역의 실행을 통해 유사성 검색 도구를위한 사용자 가이드와 함께 시작합니다. 다음으로, 우리는 진화의 가장 적합한 모델을 결정하기 위해 소프트웨어를 사용하는 프로토콜에 따라 여러 순서 정렬을 생성하기위한 프로그램을 탐구한다. 우리는 최대의 가능성과 베이지안 기준을 통해 계통 발생 학적 관계를 재구성 프로토콜 개요 마지막으로 계통 발생 수를 시각화하기위한 도구에 대해 설명합니다. 어떤 계통 발생 학적 접근 방법의 완전한 설명을 의미하여이 아니지만, 그것은 실제 시작 INFORMAT으로 독자를 제공한다일반적으로 phylogeneticists으로 활용 키 소프트웨어 응용 프로그램에 이온. 이 기사에 대한 비전은 계통 발생 학적 연구에 착수 연구자를위한 실습 도구로 봉사하고 또한 교실이나 교사 실험실에 통합 될 수있는 교육 자원으로 역할을 할 수 있다는 것입니다.

쿼리에 유사성을 발견하는 것은 연구자가 새로운 순서에 잠재적 인 정체성을 돌리는 또한 시퀀스 사이의 관계를 유추 할 수 있습니다. BLAST 1의 파일 입력 유형은 FASTA 포맷의 텍스트 순서 나 GenBank 액 번호입니다. FASTA 형식의 시퀀스는 &quot;&gt;&quot;기호 (그림 2)에 의해 표시되는 설명 라인으로 시작한다. 설명은 &quot;&gt;&quot;기호 순서 (예. 뉴클레오티드 또는 아미노산) ?...

Watch this Scientific Journal Video about A Practical Guide to Phylogenetics for Nonexperts at JoVE.com

A Practical Guide to Phylogenetics for Nonexperts

여기에서 우리는 뉴클레오티드 또는 아미노산 서열 데이터 세트에서 안정적인 계통 발생을 생성하기위한 단계별 파이프 라인을 설명합니다. 이 가이드는 연구자 또는 계통 발생 학적 분석에 새로운 학생 서비스를 제공하는 것을 목표로하고있다.

비전문가를위한 계통 발생에 대한 실용 가이드

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

a-practical-guide-to-phylogenetics-for-nonexperts

Research

JoVE Journal

Biology

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

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

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

작업 워킹 레더 렁 : 채점 시스템과 실용적인 프로그램입니다.

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

연구

생물학

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

유전자 Inducible의 운명 매핑에 대한 실용적인 접근법 : 셀을 선택하고 추적하는 비주얼 가이드 VIVO에서</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

플라즈마 단백질 보존을위한 혈액 수집 장비로부터 게놈 DNA를 추출하는 실제적이고 새로운 방법

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

서부 블로팅에서 실용적이고 편리하며 신뢰할 수 있는 토탈 단백질 로딩 제어를 위한 V3 얼룩 없는 워크플로우

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

문제 해결 전략 현대 양적 형광 웨스턴 블로 팅에 가이드

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the avoidance of airway obstruction, prevention of gastric tympany, and to allow ventilatory support. Due to the difficulty of intubation, alternative methods such as the use of laryngeal mask airways or laryngeal tubes have been explored. However, these methods do not result in direct access to the trachea and thus may present a risk for development of complications. In addition, lack of direct intubation of the trachea can result in personnel exposure to waste anesthetic gases. Numerous methods for endotracheal intubation have been described, including blind placement, use of a fiberoptic laryngoscope or endoscope, and cricoid placement. Despite these numerous publications, many still struggle to achieve success. Here we provide a detailed description of an intubation technique that can be taught with minimal training with a short time to proficiency. Briefly, after administration of injectable anesthesia and proper positioning of the rabbit, a polypropylene catheter is placed into the trachea by direct visualization using a laryngoscope. The catheter is then used as a guide to direct the endotracheal tube into the trachea. This method allows for intubation without the need for expensive equipment and can be performed by a single individual without the need for an assistant. In conclusion, this technique can be easily taught and performed at very little cost in any clinical or research setting.

Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting.

폴 리 프로필 렌 가이드 카 테 터를 사용 하 여 토끼의 endotracheal 삽 관 법

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA 방해의 실용적인 사용: Liposome 캐리어 바퀴벌레에 대 한 이중 가닥 RNA의 구두 납품

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

싱크로트론 마이크로 CT를 위한 고처리량 피질 뼈 샘플 조달 및 분석을 위한 단면, 코링 및 이미지 처리 가이드

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

모기 전기 조영술에 대한 단계별 가이드

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

난소절제술(Ovariecomized) 마우스 모델(Developing an Ovariectomized Mouse Model with Elevated FSH Level)

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

저자 스포트라이트: FSH와 병태생리학적 변화의 관계 조사(Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women) - 생쥐 모델의 통찰력 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.