우리는 화학 과학, Geosciences 및 Biosciences 부문, 기초 에너지 과학, 과학의 사무실, 에너지의 미국과 (보너스 번호 DE-FG02-91ER20021) 및 국립 과학 재단 (MCB 0,948,584) (FB)의 사무실에 의해 지원을 인정합니다. 우리는 원고를 편집 미스 카렌 버드에 감사합니다.

1. EMS 치료 Arabidopsis thaliana의 씨앗은 T / 돌연변이 5-7로 C / G 그 결과 게놈 C - 투 - T 변화로 유도 mutagen 에이전트 에틸 메탄 산염 (EMS) 3,4로 사용 mutagenized됩니다. <ol><li> organelle 형광 마커를 (특히, 본 연구에서는 ssGFPHDEL는 (신호 시퀀스-GFP-HDEL tetrapeptide) ER 마커로 사용되었습니다) 운반 0.8 g Arabidopsis 종자를 (~ 40,000 씨앗) 무게. </li><li> 50 ML 팔콘 튜브의 씨앗을 전송 후 25 ML 증류수를 추가합니다. </li><li> 0.2 % (V / V) 에틸 메탄 산염을 추가합니다. </li><li> 낮은 속도로 믹서를 nutating 16 번 시간 동안 알을 품다. </li><li> 액체를 대기음 및 1.0 M NaOH는 EMS를 inactivate이 포함된 플라스크에 그 파일을 삭제. </li><li> 가까이, 씨앗을 포함 팔콘 튜브 25 ML 물을 추가하고 5 번 반전을 씨앗을 씻어, 모든 씨앗이 정착 때까지 기다려 다음, 1.0 M 나에 물을 대기음 및 폐기<html> 플라스크, 오하이오. </li><li> 최대 10 배까지 세정 단계를 반복합니다. </li><li> 최종 세척 후에 물을 최소한의 금액에 씨앗을 다시 일시 중지합니다. </li><li> 10 % 표백제의 25 ML 추가 종자 살균을 진행, 30 s에 대한 노골적인 악수. 다음 표백제를 부어 및 멸균 물의 25 ML와 린스, 씨앗 하단으로 정착합시다. 멸균 물을 붓고 25 ML 70 % 에탄올을 추가합니다. 30의 위해 튜브를 흔들어. 씨앗이 하단으로 정착합시다. 에탄올을 붓고 멸균 물의 25 ML와 린스. 다음 3 MM 여과지를 포함하는 플라스틱 배양 접시에 씨앗을 붓다, 그리고 후드를 건조하게 두 번 멸균 물로 세탁을 반복합니다. 2 일간 4 ° C에서 저장. </li><li> ½ MS phytagel (Murashige와 Skoog 매체의 절반 농도), 150 mm 페트리 접시 (접시 ~ 250-300 씨앗)에서 플레이트 씨앗. </li><li> 2 주 동안 접시에 M1 씨앗을 재배 후 토양에 이식. </li><li> M2 라인 (1000 독립 라인) 생성 개별 M1에서 M2 식물 종자를 수집합니다. </li></ol>M2와 M3 인구의 ove_title &quot;&gt; 2. 공촛점 상영 이 섹션에서 우리는 앞서 8 설명된대로 공촛점 또는 형광 현미경과 모종의 관찰에 대해 설명합니다. <ol><li> 각 M2 라인에서 육십 종자는 플라스틱 배양 접시에 7-10일, ½ MS phytagel 위해 재배되며, 동일한 접시에도 (제어) EMS-경과 다섯 모종을 재배하고 있습니다. </li><li> 5 ~ 10 cotyledons는 현미경 슬라이드에 렌즈 (40X) 방면 abaxial 측면으로 탑재하고 coverslip과 함께 동봉합니다. </li><li> 각 떡잎은 organelle 마커의 변경된 subcellular 분포에 대한 형광 아래 피질에서 중간 영역에, 관찰됩니다. </li><li> 긍정적인 식물은 토양에 이식하고 씨를를 모으고 M3 세대에서 돌연변이 표현형 확인을 위해 다시 검사합니다. </li><li> 아마도 공동 야생 형 유전자에 backcrossing 통해 배경 변이 오염 것은 적어도 세 번 제거원하는 organelle 형광 마커를 ntaining. <ol><li> 어머니는 공장에서 미세 가위 또는 집게를 사용하면 성숙 siliques을 제거하고, 개방 꽃. </li><li> 분열 조직에서 너무 작은 봉오리를 제거합니다. </li><li> 꽃잎이 한 꽃의 꽃봉오리를 열고 모든 anthers을 제거하는 sepals 사이 포셉 중 한 쌍의 팁을 넣습니다. </li><li> 포셉 한 켤레를 사용하면 아버지는 공장에서 열린 성숙한 꽃을 가지고 emasculated 식물의 오명에서 anthers를 문지 르세요. </li></ol></li></ol> 3. 매핑 이 섹션에서는 전통적인 매핑 방법 10,11 비교한다면 빠른 Borevitz 9 일부터 수정된 프로토콜을 사용하여 열성 돌연변를 매핑하는 근본적 방법을 설명합니다. 이 접근법은 하나의 분석 12 다양한 단일 기능 polymorphisms (SFPs)을 감지하는 능력과 고밀도 oligonucleotide 어레이를 사용합니다. Affymetrix의 Arabidopsis ATH1의 GeneChip 배열을 사용하면 가능합니다약 24,000의 유전자를 분석합니다. 변이를 보여주는 F 2 개인 수영장은 같은 segregating 인구 내에서 수집된 야생 - 타입 F 2 식물의 수영장과 비교됩니다. 그런 다음 돌연변이 돌연변이 수영장은 돌연변이 유전자형의 대립 유전자에 대한 풍부하고있는 지역에 매핑되며 결과적으로 같은 지역에서 야생 형 풀은 야생 형 부모 대립 유전자 13 풍부 해집니다. <ol><li> 게놈 DNA (3 μg)은 Qiagen DNeasy 공장 미니 키트를 사용하여 homozygous 돌연변 컬럼비아 (M3)에서 추출되며 Illumina 게놈 분석기 II (GA II) 14 시퀀싱을 위해 제출합니다. </li><li> homozygous 돌연변 컬럼비아 (M3)은 매핑 인구를 생성하는 Landsberg erecta으로 건너됩니다. </li><li> 매핑은 탈선 표현형 및 야생 형 표현형와 F2를 식물의 같은 숫자를 보여주는 70 최대 100 F2를 개인에 수행됩니다. </li><li> 홀 펀치를 사용하여 잎 디스크 각 공장 (0.60 ㎜)에서 수집합니다.잎 디스크의 DNA와 유사한 금액을 가지고해야하는 같은 시대의 잎사귀로부터 수집되어야한다. 샘플은 게놈 추출 별도로 또는 그룹에 대해 처리할 수 있습니다. 이 경우, 그것은 결국 당신은 게놈 DNA를 추출되어야한다있는 적은 eppendorf 튜브가되도록 각 Eppendorf 튜브에 대해 5-10 샘플을 수집할 수 있습니다. </li><li> MasterPure 식물 잎의 DNA 정제 키트 (Epicentre)은 게놈 DNA를 추출하는 데 사용됩니다. 각 시료로부터 얻은 게놈 DNA가 nanodrop로 계량합니다. </li><li> 각 샘플의 게놈 DNA의 동일한 수량 그러면 식물 DNAs 300 NG (~ 30 μL)의 합계를 함께 넣어되며 60 μL 2.5X 무작위 primers 솔루션을 추가 [125 MM 트리스-HCL (산도 6.8), 12.5 MM MgCl2, 25 MM 2 - 메르 캅 토 에탄올은 750 μg / ML oligodeoxyribonucleotide의 primers (무작위 octamers] Bioprime 키트) 42 μL 물 (최종 부피 132 μL). </li><li> 변성의&gt; 95시 DNAs ° C에서 5-10 분. </li><li> 얼음 쿨. </li><li> 각 denatu으로붉은 DNA는 비오틴 dCTP [1 MM 비오틴-14-dCTP, 1 ㎜ dCTP, 2 MM dATP, 2 개의 MM dGTP, 10 MM 트리스-HCL 2 밀리미터 dTTP (산도 7.5), 1 ㎜ Na2EDTA]와 15 μL 10X dNTPs 믹스를 추가 (Bioprime 키트), 3 μL의 Klenow 효소 (Bioprime 키트)를 완료합니다. </li><li> 25 ° C.에 하룻밤 사이에 품어 </li><li> 믹스, 그 다음날, 15 μL 3 M NaOAc 400 μL 차가운 백퍼센트 EtOH를 추가합니다. </li><li> 1 시간 -80 ° C에서 부화 후 15 분에 해당하는 20,500 XG에 스핀, 뜨는 제거, 500 μL 감기의 75 % EtOH로 씻는다. </li><li> 10 분 동안 20,500 XG에 봐. </li><li> 100 μL 물에 10 분 및 resuspend위한 37 ° C에서 건조 DNA의 알약. </li><li> 겔 (그림 2)에서 수확량과 품질을 확인하는 5 μL를 사용합니다. </li><li> 를위한 야생 유형 및 돌연변이 GeneChip의 Arabidopsis ATH1의 게놈 배열 하이브 리다이 제이션에 대한 반응을 레이블링의 95 μL를 보냅니다. </li><li> R 소프트웨어 (사용하는 배열을 스캔하고 취득. CEL 파일을 분석하고 <a href="http://www.r-project.org/">http://www.r-project.o을</a>RG /). </li><li> R이 소프트웨어를 설치 후 프로그램을 열고 다음과 같은 문자열을 붙여 넣습니다 : 소스 ( &quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;) biocLite () 그런 다음 리턴 키 이것이 표준 Bioconductor 패키지 (설치 누르십 <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ). </li><li> 바탕 화면에 새 폴더에서 다음 웹사이트 (에서 다운로드 <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ) 파일 : readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip 파일 ColLerCEL.zip은 풀었 및 결과 파일은 새 폴더에 붙여넣어야합니다. </li><li> wildtype.CEL 및 mutant.CEL에 GeneChip 실험에서 얻은 데이터를 바꿉니다. </li><li> 귀하의 GeneChip 데이터를 지금 복사 (wildtype.CEL과해당 폴더로 mutant.CEL). </li><li> 오픈 R, Mac 용)을 누른 다음 &quot;기타&quot;그리고 &quot;변경 작업 디렉토리&quot;를 클릭하십시오. b)는 PC 용 : 클릭 &quot;파일&quot;다음 &quot;변경 디렉토리&quot; </li><li> Mac 용) :이 파일이 포함된 폴더를 선택합니다. &quot;작업 영역&quot;, &quot;로드 작업 영역 파일을 클릭하십시오 :&quot;다음 &quot;파일,로드 작업 공간&quot;을 &quot;PC를 선택 ath1V5.RData, B)을 위의 파일이 들어있는 폴더를 선택하고&quot;및 ath1V5.RData를 선택합니다. </li><li> 오픈 readCEL.R는 메모장을 사용하고 R.에 대한 전체 텍스트를 복사 </li><li> 오픈 SFP.R는 메모장을 사용하고 R.에 대한 전체 텍스트를 복사 </li><li> 오픈 Map.R는 메모장을 사용하고 R.에 대한 전체 텍스트를 복사 </li><li> 콘솔 창에서는 다음과 같은 메시지가 나타납니다 : 염색체 X는 MB yz을 제한 테어의 검색 유전자는이 링크를 붙여 넣습니다 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>&amp; 끝 = Z </li><li> 인터넷 브라우저에 링크를 복사하여 붙여 넣습니다. 창이 표시하고 파일을 열거나 저장 물을 것이다, 당신은 저장을 선택합니다. 파일 이름을 선택하고 확장자를 첨부. &quot;XLS는&quot;다음 저장을 누릅니다. </li><li> 파일을 열고 당신의 돌연변이에 대한 좌표를 (결과가 그래픽으로 그림 3에 표시됩니다) 찾을 수 있습니다. &quot;XLS&quot;. </li><li> 조립 Illumina의 매핑된 영역에서 돌연변이 게놈의 읽습 단일 염기 polymorphisms 중 특정 EMS 전환 할 4 식별합니다. 표준 절차 15을 사용하여 야생 형 유전자 (들)과 변형에 의해 돌연변이 표현형을 보완. </li></ol> 4. 대표 결과 그림 1은 공촛점 현미경 검사를 사용하여 분비 경로의 돌연변이의 식별에 사용되는 방식을 보여줍니다. 그림 2는 배열 하이브 리다이 제이션을위한 분류 게놈 DNA의 일반 준비를 보여줍니다. 그림 3, GeneChip의 Arabidopsis ATH1의 게놈 배열에서 얻은 데이터가 제공됩니다 분석하여 예상 전형 결과에서. <img alt="그림 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> ssGFPHDEL (ER 마커) (1) 표현 그림 1. Arabidopsis 형질 전환 식물은 EMS 치료 (2) 충분한 씨앗을 생산하기 위해 재배되었다. EMS로 치료 씨앗 후 M1 식물 (3)을 생성 sown되었다. 각 M1 식물은 다른 라인을 대표하고 씨앗이 각각 별도로 수집되었다. M2 씨앗이 ½ MS 기판 (4)에 도금 후 공촛점 현미경 (5)에 의해 응급실의 형태학의 결함에 대한 상영되었다. 심사하는 동안 우리는 야생 형 응급실의 형태 (6) 및 결함 응급실 phenotypes (7)를 표시 식물을 보존하는 식물을 발견. 이 공장이 M3 세대를 얻기 위해 그리고 표현형 (8)를 확인하기 위해 성장했다. M3 공장에서 게놈 DNA가 Solexa Illumina 시퀀싱 ()에 사용되었다. 같은 공장또한 F2 매핑 인구 (B)을 구하는 데 Ler-wt있는 십자가를 위해 사용되었다. <img alt="그림 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> 그림 2. Bioprime 무작위 분류 반응 (100 μL의 5 μL)는 아가로 오스 겔 1 %로드되었습니다. 레인은 야생 형 F2 식물 풀에서이며, 차선 B는 돌연변 F2 식물의 풀에서입니다. (마커 코에서 1 KB DNA를 사다리-N3232입니다.) <img alt="그림 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> 그림 3. 그림 GeneChip의 Arabidopsis ATH1 게놈 배열 하이브 리다이 제이션을 사용하여 열-0 변이의 매핑의 예를 나타냅니다. 이 예제에서 변이는 수직 막대기에 의해 염색체 1, 구분된에 있습니다. 가로 막대는 검출을위한 임계값을 나타냅니다.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+methanesulfonate-induced+reversion+of+bacteriophage+T4rII+mutants.">Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants.</a> Genetics. 48, 561-580 (1963).</li><li>Kovalchuk, I., Kovalchuk, O., Hohn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+variation+of+the+somatic+mutation+frequency+in+transgenic+plants.">Genome-wide variation of the somatic mutation frequency in transgenic plants.</a> The EMBO journal. 19, 4431-4438 (2000).</li><li>Boulaflous, A., Faso, C., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+Golgi+apparatus:+from+imaging+to+genes.">Deciphering the Golgi apparatus: from imaging to genes.</a> Traffic. 9, 1613-1617 (2008).</li><li>Borevitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+and+mapping+with+high-density+oligonucleotide+arrays.">Genotyping and mapping with high-density oligonucleotide arrays.</a> Methods in molecular biology. 323, 137-145 (2006).</li><li>Konieczny, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+mapping+Arabidopsis+mutations+using+co-dominant+ecotype-specific+PCR-based+markers.">A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers.</a> The Plant journal : for cell and molecular biology. 4, 403-410 (1993).</li><li>Bell, C. J., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+30+microsatellite+loci+to+the+linkage+map+of+Arabidopsis.">Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.</a> Genomics. 19, 137-144 (1994).</li><li>Hazen, S. P., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+arrays+are+not+just+for+measuring+gene+expression.">Gene arrays are not just for measuring gene expression.</a> Trends in Plant Science. 8, 413-416 (2003).</li><li>Hazen, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+array+mapping+of+circadian+clock+and+developmental+mutations+in+Arabidopsis.">Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.</a> Plant Physiology. 138, 990-997 (2005).</li><li>Bentley, D. 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우리는 공개 할게 없다.

여기 endomembrane의 돌연변이의 식별을 위해 공촛점 현미경 기반 검사를 설명했다. 이 접근법은 쉽게 특정 형광 단백질 마커를 사용할 수있는 세포의 다른 organelles로 연장하실 수 있습니다. 화면이 대상 organelle이나 마커를 포함해야하지 않는 organelles로 중 형광 마커의 탈선 분포를 보여 돌연변이의 신분에 따라 달라집니다. 각각 이러한 돌연변 마커를 subcompartimentalize하기 organelle의 능력 중 하나가 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Ethylmethane의 산염 </td><td> 시그마 </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT 베이커 </td><td> 3722-05 </td></tr><tr><td> Murashige skoog 기초 매체 w gamborg 비타민 </td><td> 피토 technolog laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td> 시그마 </td><td> P8169-1kg </td></tr><tr><td> RNeasy 미니 키트 </td><td> Qiagen </td><td> 74,104 </td></tr><tr><td> 마스터 순수 식물 잎의 DNA 정제 키트 </td><td> Epicentre </td><td> MPP92100 </td></tr><tr><td> Bioprime의 DNA 라벨링 시스템 </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> 알콜 200 증명 </td><td> Decan 실험실 INC. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT 베이커 </td><td></td></tr><tr><td> 유전자 칩 Arabidopsis의 ATH1게놈 배열 </td><td> Affymetrix </td><td> 900,385 </td></tr><tr><td> 팔콘 튜브 50 ML </td><td> 코닝 </td><td> 430,290 </td></tr><tr><td> Eppendorf 튜브 1.5 ML </td><td></td><td></td></tr><tr><td> 90mm 필터 종이 </td><td> 워트 먼지 </td><td> 1,001,090 </td></tr><tr><td> 분석 잔액 </td><td> Mettler 톨레도 AB54-S </td><td> NA </td></tr><tr><td> Nutating (웨이브) 흔드는 </td><td> Heidolph polymax 1040 </td><td> NA </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf 5417-R </td><td> NA </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

이 프로토콜은 Arabidopsis 모종의 형광 현미경 기반 검사를 설명하고 분비 경로에서 특정 태그를 형광 마커의 subcellular 분포를 변경 열성 돌연변를 매핑하는 방법에 대해 설명합니다. Arabidopsis 때문에 자사의 게놈 크기의 유전자 연구를위한 강력한 생물 학적 모델이며, 생성 시간, 그리고 왕국 간의 분자 메커니즘의 보존. 분자 마커를 기반으로 전통적인 방법과 대안에 변이를 매핑하는 방식으로 genotyping 배열은 비교적 빠른 있기 때문에 유리하고 아주 짧은 기간에 여러 돌연변이의 매핑을 허용할 수 있습니다. 이 방법은 식물의 어떤 organelle의 무결성에 영향을 미칠 수있는 단백질의 식별이 가능합니다. 여기 예로, 우리는 endoplasmic reticulum (ER)의 무결성을위한 중요한지도 유전자에 대한 화면을 제안합니다. 우리의 접근법은 그러나, 쉽게 다른 식물 세포 organelles로 연장하실 수 있습니다(예를 들어 1,2 참조), 따라서 다른 subcellular 구조를 지배하는 분자 기초를 이해 향한 중요한 단계를 나타냅니다.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

세포 생물학의 기본적인 탐구는 진핵 세포를 만드는 organelles의 신원을 기초 메커니즘을 정의하는 것입니다. 여기 형광 현미경과 차세대 시퀀싱 도구를 사용하여 식물 organelles의 형태학의 기능적인 무결성에 대한 책임 유전자를 식별하는 방법을 제안합니다.

형광 - 현미경 검사와 차세대 장면 : Organelle 무결성에 관련된 유전자의 식별을위한 유용한 도구

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

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JoVE Journal

Biology

12.2K 조회수.  Michigan State University. 세포 생물학의 기본적인 탐구는 진핵 세포를 만드는 organelles의 신원을 기초 메커니즘을 정의하는 것입니다. 여기 형광 현미경과 차세대 시퀀싱 도구를 사용하여 식물 organelles의 형태학의 기능적인 무결성에 대한 책임 유전자를 식별하는 방법을 제안합니다.

비디오: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

연구

생물학

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

선충류의 바디 크기 규정에 참여 유전자에 대한 화면으로 RNA로 인한 간섭 수유 전략을 사용하여<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16S 리보솜 RNA 유전자 증폭 산물의 차세대 시퀀싱

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA 메틸화 분석 차세대 시퀀싱 대상

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

다음-세대 16S rRNA Amplicon 시퀀싱에 의해 틱 미생물 특성

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2단계 PCR 및 차세대 16S rRNA 유전자 시퀀싱을 사용한 미생물 분석

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

차세대 시퀀싱을 위한 자기 비드 기반 모기 DNA 추출 프로토콜

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.