Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

PCR에 의한 디그 옥시 제닌 dUTP를 1. 레이블 프로브 <ol><li> 이전 13 설명 ​​된대로 10 배의 dNTP 믹스 디그 옥시 제닌 dUTP를을 포함하여 PCR 라벨링을 수행합니다. </li><li> 제조자의 지시에 따라 스핀 컬럼으로 정제 PCR 생성물을 정제 하였다. <ol><li> 프로브가 짧을 200 bp의 경우, 반응 혼합물로부터 스핀 - 컬럼 크로마토 그래피보다는 스핀 칼럼 정제 무료 디그 옥시 제닌 dUTP를 제거. </li></ol></li></ol> 2. 준비 폴리 라이신 코팅 슬라이드 참고 : 전체 절차는 약 2 시간 소요됩니다. 대부분의 단계는 건조 단계를 제외하고, 실온에서 수행된다. <ol><li> 슬라이드를 청소 <ol><li> 플라스틱 용기에 슬라이드를 배치하고 간단히 증류수 (DW)와 슬라이드를 헹군다. 물을 제거하고 1 % DW 유리 세정제를 함유하는 용기를 채운다. </li><li> 실온에서 50 rpm으로 (RT)에서 15 분 동안 교반 슬라이드. DW 3 슬라이드를 씻으RT에서 5 분마다 시간. </li><li> 교반과 함께 15 분 동안 70 % 에탄올로 슬라이드를 씻으십시오. 70 % 에탄올을 취소하고 15 분 동안 65 ° C 건조 블록 공기 건조에 슬라이드를 배치합니다. </li></ol></li><li> 멀티 웰 유리 슬라이드 리신 코팅 참고 : 염색 과정에 걸쳐 샘플 밀착성을 제공하기 때문에, 슬라이드 유리 라이신 코팅은 중요한 단계이다. 저조한 코팅 된 슬라이드 샘플의 손실이 발생할 것입니다. <ol><li> 0.01 % 폴리 리신으로 원액을 희석 (W / V) 증류수이다. 희석 된 0.01 %의 20 μl를 깨끗한 유리 슬라이드의 우물에 (w / v)의 폴리 리신을 추가합니다. </li><li> RT에서 5 분 동안 유리 슬라이드 부화. </li><li> 1 시간 동안 65 ° C 건조 블록 공기 건조에 슬라이드를 놓습니다. </li><li> 먼지 무료로 상자에 폴리 라이신 슬라이드를 저장합니다. </li></ol></li></ol> 슬라이드 유리에 벌레 3. 고정 (그림 1) <ol><li> FISH를위한 준비 배아 참고 : 수확 벌레를 모두 t 전에그 세균 음식 현미경 성장 배지를보고에 의해 소비된다. 기아는 성인 웜의 계란 생산을 줄이고 달걀 부화를 증가시킨다. 자세한 방법은 14, 15에 설명되어 있습니다. <ol><li> 표준 방법 (14)에 따라 50mm 페트리 접시에 벌레를 성장. </li><li> 모든 세균 음식을 섭취 한 후 주걱으로 분기 한천 미디어를 잘라. 오염을 방지하기 위해서 절단 전에 멸균 주걱. </li><li> 100mm 선충류 성장 미디어 (NGM) 접시에 한천의 모든 조각을 넣어. 신선한 박테리아 음식에 도달하는 벌레를 거꾸로 한천 조각을 돌립니다. </li><li> 48 시간 72 후, M9 버퍼와 벌레를 수집합니다. NGM 플레이트에 M9 버퍼 5 ㎖ - 3을 추가합니다. NGM의 표면에 피펫 M9 버퍼는 벌레를 씻어. </li><li> 벌레와 액체를 수집하고 15 ML 튜브에 추가 할 수 있습니다. 참고 : 30 % 자당 용액에서 수확, 원심 분리기 벌레 후 한천 이물질이있는 경우. 파편이 펠렛 동안, 웜에 떠표면. </li><li> 15 ml의 볼륨을 만들기 위해 M9 버퍼를 추가합니다. 3 분 300 XG에서 원심 분리하여 벌레를 펠렛과 M9 버퍼의 대부분을 제거합니다. 이 단계를 2 번 더 반복합니다. 참고 : 웜은 아직 원심 분리 한 후 부동 경우, 원심 분리기의 브레이크 레벨 = 1 값으로 설정. </li><li> 기음 M9 버퍼입니다. 벌레에 표백 솔루션을 추가합니다. 웜의 0.5 ml의 당 5 M KOH의 6.5 DW ml의, 차아 염소산 2 ㎖, 1 ml에 추가 할 수 있습니다. </li><li> 50 rpm에서 3 분 동안 실온에서 락으로 벌레를 품어. 15 초 동안 소용돌이 웜은 기계적으로 벌레를 전단과 계란을 노출합니다. </li><li> 표백하는 동안 해부 현미경 튜브를 관찰한다. 벌레가 반으로 잘라하고 계란이 해제되어 있는지 확인합니다. 성인 신체의 대부분이 용해되면, 15 ml의 볼륨을 만들기 위해 M9 버퍼를 추가합니다. </li><li> 3 분 동안 300 XG에 원심 분리기. 기음 M9의 대부분을 15 ml의 볼륨을 보충하기 위해 신선한 M9 버퍼를 추가합니다. 반복 세척 단계 3 회. 노트:그들은 표백 과정을 방해으로, 웜의 과도한 양을 사용하지 마십시오. 보다 8 분 세척까지 전체 반응 시간을 유지합니다. 오버 표백 계란 강한 자기 형광을 생산하고 있습니다. </li><li> 200 ㎕의 2 % PFA을 4 % 파라 포름 알데히드 (PFA) 200 μL까지 폴리 소르 베이트 20 (PBST)을 인산염 완충 식염수를 추가한다. 주의 : PFA는 발암 물질이기 때문에, PFA를 사용하기 전에 보호 복, 보호 장갑 및 눈 방패를 착용하십시오. </li><li> 폴리 라이신 코팅 된 슬라이드의 우물에 2 % PFA에서 계란의 40 μl를 추가합니다. </li><li> 습기 챔버에 슬라이드를 넣고 실온에서 15 분 동안 품어. 슬라이드는 내부에 배치 된 직후 습기 챔버를 닫습니다. 참고 : 고정 된 상태로 계란은 슬라이드의 바닥에 정착. </li></ol></li><li> 물고기를 해부 생식선 준비 <ol><li> 수확 성인 웜 M9 버퍼 1 ㎖를 피펫 팅에 의해 50mm NGM 판에 성장. 세균성 식품 전에 수확 웜은 고갈된다. </li><li> 어떤 세균 기지에서 벌레를 씻어H M9 버퍼 2 회. 참고 : 잔류 박테리아는 폴리 라이신 코팅 된 슬라이드에 달라 붙는 해부 생식선을 방해 할 수 있습니다. </li><li> 300 x g에서 원심 분리하여 벌레 펠렛. M9를 제거하고 마이크로 피펫에 의해 빈 NGM 플레이트로 웜을 전송합니다. </li><li> 폴리 라이신 처리 된 슬라이드의 잘에 2 밀리미터의 levamisole을 포함하는 M9 버퍼의 30 μl를 추가합니다. </li><li> 1 ML 튜브에 M9 버퍼의 500 μl를 추가합니다. 입 피펫으로 웜을 전송하기 위해 버퍼를 사용합니다. </li><li> M9 버퍼를 함유하는 1 ㎖의 튜브에 모세관을 배치하여 M9 완충액 입 피펫의 끝을 채운다. </li><li> 웜의 머리가 입 피펫을 입력하도록 해부 현미경, 단지 성인 웜 드래그 입 피펫의 머리 앞에 입 피펫의 팁을 넣어. 웜의 머리 끝을 입력하면, 웜의 몸 전체가 팁으로 그려집니다. </li><li> 입 피펫을 사용하여 폴리 라이신 코팅 된 슬라이드로 웜을 전송합니다. </li><li> 의 절단, 면도칼을 사용하여머리 또는 슬라이드 웜의 꼬리의 끝 (F). 웜이 절단 될 때, 생식선 밖으로 나타납니다. 생식선은 슬라이드에 충실 할 것이다. <ol><li> 하나 잘 적어도 30 벌레를 준비합니다. 추가로 30 웰 웜에 사용될 수있다. </li></ol></li><li> 습기 챔버에 슬라이드를 넣고 입으로 피펫으로 M9 버퍼를 대기음. </li><li> 습 챔버 내에서 15 분 동안 실온에서 2 % PFA 20 μl를 첨가하여 샘플을 고정한다. </li></ol></li></ol> 4. 고정 및 Permeabilization <ol><li> 드라이 아이스에 알루미늄 블록을 놓고 깊은 냉동고에 보관 (-80 ° C). -20 ° C에 보관 메탄올 및 아세톤. </li><li> PFA 고정 단계 3.1.15 또는 3.2.11 후 ~ 정착액의 5 μl를 마이크로 피펫 떠나는를 사용하여 정착액을 제거합니다. </li><li> 샘플 슬라이드에 다른 폴리 라이신 코팅 된 슬라이드를 넣습니다. 솔루션이 과도한 경우 종이 타월로 정착액을 제거합니다. 그들은 서로 붙어되면 슬라이드를 이동하지 마십시오. </li><li> 슬라이드를 고정적어도 15 분 동안 알루미늄 블록. 주의 : 샘플을 적어도 2 저장 될 수있다 - 삼일. </li><li> 슬라이드가 고정되는 동안 얼음 냉 메탄올 및 아세톤을 함유하는 병을 넣어. </li><li> 슬라이드를 꺼내 샘플을 동결 균열을 비틀어. 상단 슬라이드를 폐기하십시오. 즉시 5 분간 빙냉 메탄올로 슬라이드 소크. </li><li> 5 분 동안 얼음처럼 차가운 아세톤으로 슬라이드를 전송합니다. </li><li> 잔류 정착액을 제거하기 위해 5 분 동안 슬라이드를 PBST 3 회 반복한다. 다음 단계로 진행 또는 4 ° C에서 100 % 에탄올에 슬라이드를 저장합니다. 주의 : 샘플을 적어도 2 저장 될 수있다 - 삼일. </li></ol> 고정 세포의 5. 하이브리드 <ol><li> (PBST 10 μg의 / ㎖의 RNase을 포함)의 RNase 용액 20 μl를 추가합니다. 1 시간 동안 37 ℃에서 습한 챔버에서 슬라이드 부화. </li><li> 폴리 소르 베이트 20 (2 배 SSCT) 15 분 각각에 2 배 식염수와 구연산 나트륨에 두 번 슬라이드를 씻으십시오. </li><li> 20 추가하이브리드 솔루션의 μL와 37 ° C 배양기에서 습기 챔버를했습니다. 1 시간 후, 피펫으로 하이브리드 솔루션을 제거합니다. </li><li> 하이브리드 솔루션을 제거하기 전에 프로브를 준비합니다. 상기 프로브는 이중 쇄 DNA가되면, 건조 블록을 5 분 동안 95 ℃에서 가열에 의해 프로브를 변성. 가열 후, 얼음 짧게에 프로브를 냉각. </li><li> 샘플에 프로브를 포함하는 하이브리드 용액 10 μl를 추가합니다. 2,000 파기 표지 된 프로브 1의 비율로 사용 농도 : 200 PNA 프로브 1의 비율로 사용 농도. 커버 유리와 샘플을 커버. </li><li> 열 블록 (80 ° C)에 물을 적신 종이 타월을 넣어. 습도 및 온도를 유지하기 위해 열 블록 플라스틱 상자 덮개를 넣는다. </li><li> 가열 블록의 온도 (80 ℃로) 안정화 된 후, 가열 된 종이 수건 시료 슬라이드를 놓고 플라스틱 박스 커버와 함께 샘플을 커버한다. 3 분 동안 샘플을 변성. </li><li> Inc의하룻밤 37 ℃에서 습기 챔버에서 슬라이드를 ubate. </li></ol> 6. 세척 및 면역 형광 <ol><li> 37 ° C에 하이브리드 세척 용액 (2 × SSC, 50 % 포름 아미드)를 따뜻하게. </li><li> 5 분 동안 실온에서 두 번 PBST에서 샘플을 씻으십시오. 커버 유리를 제거합니다. </li><li> 30 분 동안 37 ℃에서 혼성화 세척 용액의 샘플을 씻는다. </li><li> 3 회 RT에서 PBST의 샘플 슬라이드를 씻으십시오. 참고 : RT에 습기 챔버의 모든 후속 단계를 수행합니다. </li><li> 차단 용액 20 μl를 추가하고 습기 챔버에서 실온에서 1 시간 동안 배양한다. </li><li> 솔루션을 차단 제거하고 FITC 접합 된 항 디곡 시게 닌 항체 용액 (1 : 200)를 추가 RT 또는 밤새 4 ℃에서 3 시간 동안. </li></ol> 7. 설치 및 관측 <ol><li> 15 분마다 PBST로 2 번 샘플 슬라이드를 씻으십시오. </li><li> DAPI와 솔루션을 장착의 10 μl를 추가합니다. 부드럽게 커버 유리하고 Enter 키를 넣습니다. 초과 솔루션을 제거종이 수건. </li><li> 장착 용액의 증발을 방지하기 위해, 매니큐어와 커버 유리의 가장자리를 밀봉. </li><li> 공 초점 현미경으로 관찰한다. 높은 배경으로 배아를 제외합니다. 20 핵 - 4 필드에 초점을 맞 춥니 다. </li><li> 100X 대물 렌즈와 제조업체의 지시에 따라 이미지를 가져 가라. 참고 : FITC에 대한 488 nm의 레이저로, CY3에 대한 555 nm의 레이저로, DAPI에 대한 405 nm의 레이저로 샘플을 익사이트. </li></ol> 텔로미어 신호의 8 정량 참고 : 이전 16 설명한 바와 같이 정량화가 이루어졌다. 비교 될 수있는 모든 이미지는 노출 시간 및 광원을 포함 동일한 설정으로 촬영한다. <ol><li> 이 .tif 형식으로 이미지를 보냅니다. </li><li> 다운로드 및 이미지 분석 소프트웨어를 설치한다. </li><li> 이미지 분석 소프트웨어를 실행하고 버튼을 동의 클릭합니다. </li><li> 열기 버튼을 클릭합니다. 이미지 파일을 두 번 클릭하여 텔로미어의 FISH와 이미지를 엽니 다. </li><li> 딸깍 하는 소리[편집] - [선택 처리 영역, 왼쪽 버튼으로 클릭하고 드래그하여 관심의 선택 영역입니다. 모든 비특이적 염색을 제외. </li><li> [광학 밀도를 발견] 텔로미어 신호 채널을 선택하고 .txt 파일에 결과를 저장할 파일 이름을 입력 - [측정]를 클릭합니다. 주 : 결과 열은 다음 순서대로 : 스폿의 형광 반점의 스폿 영역의 배경 강도. </li><li> 값을 복사하여 현장의 형광에서 현장의 배경 강도를 뺍니다. 값은 이제 통계적 분석 될 수있다. </li></ol>

<ol>
	<li>Reddel, R. R., Bryan, T. M., Murnane, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+cells+with+no+detectable+telomerase+activity.+A+review.">Immortalized cells with no detectable telomerase activity. A review.</a> Biochemistry-Moscow. 62, 1254-1262 (1997).</li><li>Seo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+maintenance+through+recruitment+of+internal+genomic+regions.">Telomere maintenance through recruitment of internal genomic regions.</a> Nat Commun. 6, 8189 (2015).</li><li>Cesare, A. J., Reddel, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+lengthening+of+telomeres:+models,+mechanisms+and+implications.">Alternative lengthening of telomeres: models, mechanisms and implications.</a> Nat Rev Genet. 11, 319-330 (2010).</li><li>Meier, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=trt-1+is+the+Caenorhabditis+elegans+catalytic+subunit+of+telomerase.">trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.</a> Plos Genetics. 2, 187-197 (2006).</li><li>Cawthon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+measurement+by+quantitative+PCR.">Telomere measurement by quantitative PCR.</a> Nucleic Acids Res. 30, 47 (2002).</li><li>Raices, M., Maruyama, H., Dillin, A., Karlseder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+of+longevity+and+telomere+length+in+C.+elegans.">Uncoupling of longevity and telomere length in C. elegans.</a> PLoS Genet. 1, 30 (2005).</li><li>Southern, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Specific+Sequences+among+DNA+Fragments+Separated+by+Gel-Electrophoresis.">Detection of Specific Sequences among DNA Fragments Separated by Gel-Electrophoresis.</a> Journal of Molecular Biology. 98, 503 (1975).</li><li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+extension+by+telomerase+and+ALT+generates+variant+repeats+by+mechanistically+distinct+processes.">Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes.</a> Nucleic Acids Res. 42, 1733-1746 (2014).</li><li>Cheung, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+telomere+length+revealed+by+single+telomere+length+analysis+in+Caenorhabditis+elegans.">Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans.</a> Nucleic Acids Res. 32, 3383-3391 (2004).</li><li>Shtessel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+POT-1+and+POT-2+repress+telomere+maintenance+pathways.">Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways.</a> G3. 3, 305-313 (2013).</li><li>Duerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook (The C. elegans Research Community). , (2006).</li><li>Phillips, C. M., McDonald, K. L., Dernburg, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+analysis+of+meiosis+in+Caenorhabditis+elegans.">Cytological analysis of meiosis in Caenorhabditis elegans.</a> Meiosis: Volume 2, Cytological Methods. , 171-195 (2009).</li><li>Emanuel, 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=Simple+and+efficient+system+for+synthesis+of+non-radioactive+nucleic+acid+hybridization+probes+using+PCR.">Simple and efficient system for synthesis of non-radioactive nucleic acid hybridization probes using PCR.</a> Nucleic acids research. 19, 2790 (1991).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J Vis Exp. , e4019 (2012).</li><li>Poon, S. S. S., Martens, U. M., Ward, R. K., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+measurements+using+digital+fluorescence+microscopy.">Telomere length measurements using digital fluorescence microscopy.</a> Cytometry. 36, 267-278 (1999).</li><li>Ferreira, H. C., Towbin, B. D., Jegou, T., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shelterin+protein+POT-1+anchors+Caenorhabditis+elegans+telomeres+through+SUN-1+at+the+nuclear+periphery.">The shelterin protein POT-1 anchors Caenorhabditis elegans telomeres through SUN-1 at the nuclear periphery.</a> J Cell Biol. 203, 727-735 (2013).</li><li>Lee, M. H., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+in+situ+hybridization+of+dissected+gonads.">RNA in situ hybridization of dissected gonads.</a> WormBook. , 1-7 (2006).</li><li>Tabara, H., Motohashi, T., Kohara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-well+version+of+in+situ+hybridization+on+whole+mount+embryos+of+Caenorhabditis+elegans.">A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans.</a> Nucleic Acids Res. 24, 2119-2124 (1996).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescence+in+situ+hybridization+(Q-FISH).">Quantitative fluorescence in situ hybridization (Q-FISH).</a> Curr Protoc Cell Biol. 18, 14 (2001).</li></ol>

The authors have nothing to disclose.

우리 프로토콜의 주요 장점은 세포 구조의 형태로 눈에 띄는 손상없이 절차의 단순함이다. 여러 단계 C.에 최적화 된 이 프로토콜에 간스의 물고기. 성공적인 FISH를위한 중요한 단계는 프로브의 라벨, 배아 및 침투의 고정을 포함한다. 디그 옥시 제닌 표지 된 dUTP의 방법은, PCR이나 닉 - 번역에 의해 사용하기 쉬운 표시 방법을 제공한다. 긴 표적 서열 레이블을 지정하려면, 닉 번역이 ?...

텔로미어는 비정상적인 융합 저하로부터 염색체 끝 부분을 보호합니다. 포유류 텔로미어는 G-풍부한 hexameric 반복, TTAGGG 및 shelterin 단지로 구성되어있다. 선충의 텔로미어 반복 서열은 포유 동물 (TTAGGC)의 그것과 유사하다. 대부분의 진핵 세포는 염색체 말단에 텔로미어의 반복을 추가하는 텔로 머라 제를 사용한다. 그러나, 10 - 암세포의 15 % 텔로미어 (ALT) 3의 대안 길어 알려진 텔로 머라 독립 메커니즘을 이용한다. 이전에, 우리는 텔로미어의 반복과 TALT라는 이름으로 관련 시퀀스가 중요한 불임이 살아 텔로 머라 돌연변이 라인의 텔로미어에서 증폭 된 보도했다. 텔로미어 길이를 정량적 PCR에 의해 또는 전체 -4,5,6,7- 텔로미어의 평균 길이를 제공 서던 블롯에 의해 측정 하였다. 전체 게놈 시퀀싱 데이터에서 텔로미어 반복의 읽기 횟수는 총 텔로미어의 내용 (8)의 지표입니다. 죄 있지만GLE 텔로미어 길이 분석 (STELA) 단일 텔로미어의 길이가,이 텔로미어 (9)의 공간 정보를 제공 할 수 제공 할 수있다. POT-1 :: mCherry 리포터 단백질이 생체 내에서 텔로미어의 공간 정보를 제공하는 반면 POT-1 단백질 (10)을 결합 단일 가닥 텔로미어이기 때문에, 그것은 이중 가닥 말단 소립의 길이를 나타낼 수 없다. 상기 방법은 반복적 인 시퀀스의 평균 정보를 제공하는 반면, 현장 하이브리드 (FISH)의 형광 염색체 규모에 금액과이자의 개별 시퀀스의 공간 패턴을 관찰 할 수 있습니다. 대신 DNA의 정제, 조직 또는 세포는 FISH에서 기본 공간 정보를 보존하기 위해 고정되어있다. 따라서, FISH는 텔로미어 반복 개별 반복 서열의 관찰을위한 양적 및 질적 도구입니다. 이 프로토콜은 모두 telo의 동시 검출을위한 효율적인 방법을 제공단순한 및 기타 반복 이전에 기술 된 방법 (11, 12)에서 개선을 기반으로. C. 엘레 유충 또는 성인 고도로 분화 된 세포와 다세포 유기체이다. 세포의 텔로미어 이질성 스폿 다수의 정량적 분석을 방해한다. 분석 된 세포의 수를 최대화하기 위해 배아 절연 및 FISH 용 폴리 리신 코팅 된 슬라이드에 도포한다. 또한,이 프로토콜은 또한 면역와 결합 될 수있다. 프로토콜이 동작하는 증거로서, 우리는 두 가지 관찰 반복 서열을 정량화 할 수 있음을 보여준다. TALT1에 대한 DNA 프로브는 간단한 PCR은 디그 옥시 제닌-dUTP를 통합하여 생성되었습니다. 그런 다음이 TALT1 프로브와 형광 표지 텔로미어 PNA 프로브를 동시에 하이브리드 하였다. 그 후, 디그 옥시 제닌은 표준 면역 형광 방법에 의해 검출되었다. 우리는 여기 TALT1는 TRT-1의 텔로미어와 colocalized 대표 이미지를 제시 </em은&gt; 생존자.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PNA probe</td>
			<td>PANAGENE</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-Fluorescein, Fab fragments</td>
			<td>Roche</td>
			<td>11207741910</td>
			<td>use 1:200 diluted in PBST</td>
		</tr>
		<tr>
			<td>Digoxigenin-dUTP</td>
			<td>Roche</td>
			<td>11573152910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SIGMA-ALDRICH</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-6148</td>
			<td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizaiton solution</td>
			<td></td>
			<td></td>
			<td>3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA</td>
		</tr>
		<tr>
			<td>Hybridizaiton wash solution</td>
			<td></td>
			<td></td>
			<td>2X SSC, 50% formamide</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>BIONEER</td>
			<td>C-9012</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SIGMA-ALDRICH</td>
			<td>H3393</td>
			<td>make 10 mg/ml for stock solution</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>67578</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% tween-20</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-2287</td>
			<td>Commercial name is Tween-20</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution (0.1 % w/v)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8920</td>
			<td>prepare fresh 0.01 % w/v solution before use</td>
		</tr>
		<tr>
			<td>M9</td>
			<td></td>
			<td></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L</td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>20% sodium hypochlorite, 0.5 M KOH</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td></td>
			<td></td>
			<td>1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Antibody buffer with 5% bovine serum albumin (BSA)</td>
		</tr>
		<tr>
			<td>illustra Microspin G-50</td>
			<td>GE healthcare</td>
			<td>27-53310-01</td>
			<td></td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td></td>
			<td></td>
			<td>To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1 % tween-20</td>
		</tr>
		<tr>
			<td>10x digoxigenin-dUTP mix</td>
			<td></td>
			<td></td>
			<td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP</td>
		</tr>
		<tr>
			<td>PCR purification columns</td>
			<td>Cosmo genetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cleaner / ULTRA CLEAN</td>
			<td>Dukssan pure chemicals</td>
			<td>8AV721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well glass slide</td>
			<td>MP biomedicals</td>
			<td>96041205</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth media</td>
			<td></td>
			<td></td>
			<td>to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>SIGMA-ALDRICH</td>
			<td>196142</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Feather</td>
			<td>blade No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Enzynomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microsope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td>EC Plan-Neofluar 100x was used as objective lens.</td>
		</tr>
		<tr>
			<td>Dry block / aluminum block</td>
			<td>Labtech</td>
			<td>LBH-T03</td>
			<td>Set temperature to 80&#8451;</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Plastic box filled with paper towel soaked in DW</td>
		</tr>
		<tr>
			<td>Image Analysis Software&#160;</td>
			<td>Dr. Peter Landsdorp</td>
			<td>TFL-telo</td>
			<td><a href="http://www.flintbox.com/public/project/502">http://www.flintbox.com/public/project/502</a></td>
		</tr>
	</tbody>
</table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

그것은 이전에 해당 ALT 생존자는 내부적으로 &#39;ALT의 템플릿&#39;(TALT) 텔로미어 유지 보수 2 시퀀스 지역화 복제하여 낮은 주파수에서, TRT-1 (ok410), 텔로 머라 아제가 결핍 된 돌연변이 체에서 나타날 수 있습니다보고되었다. PNA 프로브를 사용하여, 우리는 해부 생식선 (도 2A)에서 텔로미어를 시각화 할 수 있었다. 희미한 텔로미어 신호에 TRT-1...

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Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

관측 및 텔로미어의 정량 및 반복 시퀀스 사용하여 형광<em&gt; 현장에서</emPNA 프로브에와&gt; 하이브리드 (FISH)<em&gt; 예쁜 꼬마 선충</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

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Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

10.3K 조회수.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

비디오: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

형광 현장에서</em인간의 정자에서> 하이브리드화 (물고기) 프로토콜

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

연구

생물학

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA 형광하여 단일 세포 유전자 전사의 분석<em&gt; 현장에서</em&gt; 하이브리드 (물고기)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

형광등과 결합 염색체 복제 타이밍<em&gt; 현장에서</em&gt; 하이브리드

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

에서 프로스타글란딘 추출 및 분석<em&gt; Caenorhabditis의 엘레</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.