Name: detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice
Uploaded: 2017-01-11T15:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

본 연구는 국립 항공 우주국 통해 아칸소 공간 그랜트 협회와 국립 우주 바이오 메디컬 연구소에 의해 지원되었다, 부여 NNX15AK32A (RP) 및 RE03701 (MH-J), 및 P20의 GM109005 (MH-J), 미국 재향 군인의 관리 ( MH-J). 우리는 원고의 준비 편집 지원 크리스토퍼 Fettes, 의료 과학 아칸소 대학의 환경 및 산업 보건학과에 대한 프로그램 코디네이터, 감사합니다.

모든 동물 연구는 국립 보건원 (National Institutes of Health)의 실험 동물의 관리 및 사용에 대한 가이드의 권장 사항을 엄격히 준수하여 수행 하였다. 동물 프로토콜은 의료 과학에 대한 아칸소 대학의 기관 동물 관리 및 사용위원회에 의해 승인되었다. 신선한 공기와 표준 설치류 음식과 물을 무료로 이용할 수의 15 시간당 사이클 - 모든 동물은 10 20 ± 2 ℃에서 표준 에어컨 동물 시설에서 보관 하였다. 도착 후, 마우스 1 주일 동안 격리에서 개최하고 인증 설치류 식사를 제공했다. 1.Radiation 노출 및 중기 세포의 생체 체포에 <ol><li> 노출 조사 챔버에서 2 Gy의 전신 방사선에 마우스를 해제는-마취. 조사 기간 동안, 통풍이 잘 유지 챔버의 장소 마우스는 자유롭게 이동하고 균일 한 용량을받지 못한 확인합니다. </li><li> 0.05 % 콜히친 용액 100 μL를 주입 (c칼슘 및 마그네슘없는 PBS에 용해 olchicine 분말) 복강 내 1 ㎖의 일회용 주사기에 부착 된 25 G 바늘을 사용. 어떤 기관에 직접 분사하지 마십시오. </li><li> 골수 세포 수확하기 전에 2 시간 동안 케이지에 동물을 둡니다. 통증이나 고통의 흔적에 대한 동물을 관찰한다. </li></ol> 2. 골수 세포 수확 및 밀도 구배 원심 분리하여 골수 단핵 세포의 분리 <ol><li> CO 2 질식하여 마우스를 안락사. </li><li> 지느러미와 복부 측면에 70 % 에탄올 스프레이. </li><li> 날카로운 가위로 복부 피부에 2cm의 절개를합니다. 무딘 핀셋 절개의 양쪽 피부를 잡고 조심스럽게 복부 근육을 잡아 당깁니다. </li><li> 가위로 양쪽 뒷다리를 잘라 즉시 4 % FBS 사전 냉장 PBS에 배치합니다. </li><li> 조심스럽게 날카로운 면도칼과면 거즈 붕대로 뒷다리에 연결된 모든 근육을 청소합니다. </li><li> 대퇴골의 경골을 분리합니다. 면도칼 각 뼈의 양쪽 끝을 낸다. </li><li> 3 mL의 주사기에 부착 된 23 G 바늘을 사용하여 (4 % FBS)와 3 mL의 PBS와 함께 골수의 컨텐츠를 플러싱하고, 15 mL의 원심 분리 튜브 내의 컨텐츠를 수집한다. 단일 세포 현탁액을 만들기 위해 바늘을 통해 세포 현탁액을 10 배 이상을 합격. </li><li> 조심스럽게 세포 현탁액 및 림프구 분리 매질 간의 인터페이스를 방해하지 않고 림프구 분리 매질 (3 mL)을 동일 부피의 세포 현탁액을 오버레이. 실온에서 30 분 동안 400 × g으로 원심 분리기. </li><li> 새로운 15 mL의 원심 분리 튜브 내의 다른 계층과 전송을 방해하지 않고 조심스럽게 버피 코트를 수집한다. </li></ol> 중기 세포 스프레드 3. 준비 <ol><li> 버피 코트를 포함하는 튜브에 10 mL의 PBS를 추가합니다. </li><li> 실온에서 5 분 동안 400 × g으로 원심 분리기. </li><li> 조심스럽게 뜨는을 제거합니다. 그런 t을 휴식그는 부드러운 도청과 펠렛 셀, ​​10 mL의 PBS를 추가합니다. </li><li> 실온에서 5 분 동안 400 × g으로 원심 분리기. </li><li> , 세포 펠렛을 방해하지 않고 뜨는을 제거 펠렛을 중단하고 미리 예열 저장성 0.075 M 염화칼륨 용액 4 mL를 넣고. 부드러운 일정한 흔들림과 강하에 의한 저장성 용액 방울을 추가합니다. </li><li> 수욕에서 37 ℃에서 20 분 동안 세포를 인큐베이션. </li><li> (: 빙초산 3 : 메탄올 1 v / v)의 저장성 처리 후, 고정액 동량 (4 ml)에 추가 15ml의 튜브와 상기 튜브를 반전시켜 부드럽게 혼합한다. </li><li> 실온에서 5 분 동안 400 × g으로 원심 분리하고 상층 액을 제거한다. </li><li> 몇 초 동안 부드러운 소용돌이 다음 탭핑과 세포 펠렛을 휴식과 일정한 흔들림와 드롭으로 정착 솔루션 드롭 3 mL를 넣고. </li><li> 실온에서 30 분 동안 배양 한 다음 5 분 동안 400 × g으로 원심 분리기. </li><li> , 상층 액을 제거 신사와 세포 펠렛을 휴식제작 : 도청, 3 mL의 신선한 정착액을 추가합니다. </li><li> 실온에서 5 분 동안 400 × g으로 원심 분리하고 상층 액을 제거한다. 부드러운 도청과 세포 펠렛을 휴식, 신선한 3 ML의 정착액을 추가합니다. </li><li> 단계를 반복 3.12 배 더. </li><li> 실온에서 5 분 동안 400 × g으로 원심 분리하고 세포 펠렛을 교란시키지 않고 상층 액을 제거한다. 그 후 600 μL의 정착에 400를 추가하고 피펫으로 잘 혼합한다. </li><li> 45 ° 각도로 기울어 미리 청소하고 젖은 슬라이드에 30 μL에게 고정 된 세포를 삭제하고 완전히 밤새 공기 건조에 슬라이드를 할 수 있습니다. </li></ol> mFISH 4. 마우스 염색체 페인팅 <ol><li> 염색체 변성 <ol><li> 실온에서 2 분 동안 2 × SSC에서 중기 세포의 퍼짐을 포함하는 슬라이드를 놓는다. </li><li> 70 %에서 2 분 각각 80 %, 100 %를 직렬 에탄올 세척하여 슬라이드를 탈수. 65 ° C에서 60 분 동안 품어. </li><li> 데우고 40 ㎖의 변성 용액 (70 % 포름 아미드 / 2 × SS기음; 30 분 동안 유리 코 플린 항아리에 70 ° C (± 2 ° C)로 pH를 7.0). 1.5 분 - 1 예열 변성 용액 슬라이드를 배양하여 염색체 변성. </li><li> 2 분 변성 과정을 중단뿐만 아니라, 재 어닐링에서 염색체 변성을 방지하기위한 빙냉 70 % 에탄올 중에 슬라이드를 즉시 급냉. 그 다음 2 분 동안 80 % 에탄올로 세척하여 탈수. 2 분 동안 100 %의 에탄올과 에탄올 세척을 반복합니다. 완전히 실온에서 슬라이드를 건조. </li></ol></li><li> 변성 및 프로브 혼합물의 하이브리드 <ol><li> 간단히 제조자에 의해 공급되는 프로브 혼합물을 원심 분리하여, 7 분 동안 수욕에서 80 ℃ (± 2 ℃)에서 배양 캡 원심 분리 튜브 스냅 500 μL로 프로브 액 10 μL를 전송하고, 변성. </li><li> 10 분 동안 37 ℃에서 수욕 프로브 혼합물 원심 튜브를 놓는다. </li><li> 변성 chromoso와 슬라이드에 변성 프로브 혼합물을 적용MES. </li><li> 조심스럽게 18mm X 18mm 유리 커버 슬립으로 영역을 커버하고 매우 부드럽게 밀어 커버 슬립을 눌러 눈에 보이는 기포를 제거한다. 고무 시멘트와 커버 슬립의 네면을 밀봉 및 하이브리드에 대한 가습 챔버에서 37 ° C에서 어둠 속에서 16 시간에 12 시간 동안 배양. </li></ol></li><li> 포스트 하이브리드 세척 및 검출 <ol><li> 조심스럽게 고무 시멘트와 커버 슬립을 제거합니다. </li><li> 5 분 동안 74 ° C (± 2 ° C)에서 미리 예열 0.4 배의 SSC의 장소 슬라이드. </li><li> 2 분 동안 세척 용액 II (4 배 SSC / 0.1 % 트윈 20)에서 세척 슬라이드. </li><li> DAPI의 Counterstain과 (1.5 μg의 / mL)로 안티 - 페이드 장착 매체의 20 μL를 놓고 유리 커버 슬립으로 다룹니다. 부드럽게 실험실 조직과 coverslip에 눌러 공기 거품과 초과 장착 솔루션을 제거합니다. 매니큐어로 커버 슬립의 가장자리를 밀봉합니다. </li><li> 적절한 필터가 장착 된 형광 현미경을 사용하여 볼 슬라이드. </li></ol></l난&gt; </ol> 5. 스펙트럼 핵형 마우스 염색체의 (SKY) <ol><li> 염색체와 프로브 변성 <ol><li> 흔들림없이, 2 분 동안 실온에서 2 × SSC에서 슬라이드 평형. </li><li> 실온에서 2 분마다 에탄올 시리즈 슬라이드 (70 %, 80 %, 100 % 에탄올)을 탈수. 완전히 에탄올을 제거 할 수있는 슬라이드를 공기 - 건조. </li><li> 따뜻한 40 ML의 변성 용액 (70 % 포름 아미드 / 2 × SSC, 산도 7.0) 30 분 동안 유리 코 플린 항아리입니다. </li><li> 1 ~ 1.5 분 동안 예열 변성 솔루션에 슬라이드를 품어. </li><li> 2 분 변성 과정을 중단뿐만 아니라, 재 어닐링에서 염색체 변성을 방지하기 위해 즉시 빙냉 70 % 에탄올로 슬라이드 젖어. </li><li> 실온에서 2 분 동안 100 % 에탄올로 2 분 후, 80 % 에탄올로 배치 의해 슬라이드 탈수. </li><li> 80 ° C (± 2 ° C)로 설정 수욕에서 SKY 프로브 (제조사에서 제공 한 유리 병 # 1) 변성7 분 후 즉시 10 분간 37 ° C로 설정 다른 수조에 놓는다. </li></ol></li><li> 프로브 하이브리드 <ol><li> 변성 염색체에 변성 SKY 프로브의 10 μL를 추가합니다. </li><li> 기포가 커버 슬립 아래에 갇혀되지 않도록 조심스럽게 SKY 프로브 상에 18mm X 18mm 유리 coverslip에 배치합니다. </li><li> 고무 시멘트와 커버 슬립의 가장자리를 밀봉합니다. </li><li> 가습 차광판 챔버에 슬라이드를 넣고 36 시간 ~ 24 시간 동안 37 ℃에서 어둠 속에서 배양한다. </li></ol></li><li> 포스트 하이브리드 세척 및 형광 검출 <ol><li> 커버 슬립을 방해하지 않고 매우 신중 고무 시멘트를 제거합니다. </li><li> 일정한 흔들림 5 분 동안 72 ° C (± 2 ° C)에서 미리 예열 빠른 세척 용액 (0.4 배의 SSC)에 슬라이드를 놓습니다. 세척 용액 III (4 배 SSC / 0.1 % 트윈 20)에 슬라이드를 담그고 흔들어 섞으면 서 1 분 동안 품어. </li><li> 옵션 : 차단 시약 80 μL를 추가하이브리드의 영역 상 (제조사에서 제공 한 유리 병 # 2)와, 24mm X 60mm 플라스틱 커버 슬립을 배치하고, 가습 실에서 30 분 동안 37 ° C에서 어둠 속에 품어. </li><li> 조심스럽게 플라스틱 커버 슬립을 제거하고 5 분간 예열 (45 ° C) 세척 용액 III와 슬라이드를 씻는다. </li><li> 80 μL Cy5에 염색 시약 (제조사에서 제공 한 유리 병 # 3)를 적용하는 24mm X 60mm 플라스틱 커버 슬립을 배치하고, 가습 실에서 40 분 동안 어둠 속에서 37 ° C에서 품어. </li><li> 함유하는 유리 코 플린 항아리에 슬라이드를 담근 예열 (45 ºC) 세정액 III (4X SSC / 0.1 % 트윈 20)와 진탕하면서 2 분 동안 수욕에서 45 ℃에서 배양한다. 이 세척 단계를 3 번 ​​반복합니다. </li><li> 80 μL Cy5.5의 염색 시약 (제조사에서 제공 한 유리 병 # 4)를 적용하는 24mm X 60mm 플라스틱 커버 슬립을 배치하고, 가습 실에서 40 분 동안 어둠 속에서 37 ° C에서 품어. </li><li> (미리 예열과 슬라이드 3 회 반복한다45 ºC) 세정액 III. </li><li> 초과 액체를 배출하기 위해 종이 타월에 대하여 기울어 진 위치에 슬라이드를 잡습니다. 20 μL 안티 페이드 DAPI 시약 (제조업체에서 제공 유리 병 # 5)를 추가하고 조심스럽게 기포를 도입하지 않고 24mm X 60mm 유리 coverslip에 배치합니다. 매니큐어로 커버 슬립의 가장자리를 밀봉 SKY 이미지를 캡처 장착 된 표면 형광 현미경으로 관찰한다. </li></ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+detection+of+RNA-DNA+hybrid+molecules+in+cytological+preparations.">Formation and detection of RNA-DNA hybrid molecules in cytological preparations.</a> Proc Natl Acad Sci U S A. 63, 378-383 (1969).</li><li>Bauman, J. G., Wiegant, J., Borst, P., van, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochromelabelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA.</a> Exp Cell Res. 128, 485-490 (1980).</li><li>Hopman, A. H., 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=Bi-color+detection+of+two+target+DNAs+by+non-radioactive+in+situ+hybridization.">Bi-color detection of two target DNAs by non-radioactive in situ hybridization.</a> Histochemistry. 85, 1-4 (1986).</li><li>Nederlof, P. 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=Three-color+fluorescence+in+situ+hybridization+for+the+simultaneous+detection+of+multiple+nucleic+acid+sequences.">Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences.</a> Cytometry. 10, 20-27 (1989).</li><li>Nederlof, P. 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=Multiple+fluorescence+in+situ+hybridization.">Multiple fluorescence in situ hybridization.</a> Cytometry. 11, 126-131 (1990).</li><li>Szeles, A., Joussineau, S., Lewensohn, R., Lagercrantz, S., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+spectral+karyotyping+(SKY)+in+biodosimetry+for+the+triage+situation+following+gamma+irradiation.">Evaluation of spectral karyotyping (SKY) in biodosimetry for the triage situation following gamma irradiation.</a> Int J Radiat Biol. 82, 87-96 (2006).</li><li>Camparoto, M. L., Ramalho, A. T., Natarajan, A. T., Curado, M. P., Sakamoto-Hojo, E. 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Res. 530, 1-7 (2003).</li><li>Edwards, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+'Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">A critique of 'Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 78, 867-871 (2002).</li><li>Bauchinger, 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=Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 77, 259-267 (2001).</li><li>Hieber, 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=Chromosomal+rearrangements+in+post-Chernobyl+papillary+thyroid+carcinomas:+evaluation+by+spectral+karyotyping+and+automated+interphase+FISH.">Chromosomal rearrangements in post-Chernobyl papillary thyroid carcinomas: evaluation by spectral karyotyping and automated interphase FISH.</a> J Biomed. Biotechnol. 2011, 693691 (2011).</li><li>Cohen, N., 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=detection+of+chromosome+rearrangements+in+two+cases+of+tMDS+with+a+complex+karyotype.">detection of chromosome rearrangements in two cases of tMDS with a complex karyotype.</a> Cancer Genet Cytogenet. 138, 128-132 (2002).</li><li>Pathak, R., 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=The+Vitamin+E+Analog+Gamma-Tocotrienol+(GT3)+Suppresses+Radiation-Induced+Cytogenetic+Damage.">The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage.</a> Pharm Res. , (2016).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, 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=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61, 387-393 (1996).</li><li>Deng, W., Tsao, S. W., Lucas, J. N., Leung, C. S., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+improving+metaphase+chromosome+spreading.">A new method for improving metaphase chromosome spreading.</a> Cytometry A. 51, 46-51 (2003).</li><li>Hande, M. P., 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=Past+exposure+to+densely+ionizing+radiation+leaves+a+unique+permanent+signature+in+the+genome.">Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome.</a> Am J Hum Genet. 72, 1162-1170 (2003).</li><li>Tug, E., Kayhan, G., Kan, D., Guntekin, S., Ergun, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+long-term+effects+of+ionizing+radiation+through+measurement+of+current+sister+chromatid+exchange+(SCE)+rates+in+radiology+technologists,+compared+with+previous+SCE+values.">The evaluation of long-term effects of ionizing radiation through measurement of current sister chromatid exchange (SCE) rates in radiology technologists, compared with previous SCE values.</a> Mutat. Res. 757, 28-30 (2013).</li><li>Kanda, R., 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=Non-fluorescent+chromosome+painting+using+the+peroxidase/diaminobenzidine+(DAB)+reaction.">Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction.</a> Int J Radiat Biol. 73, 529-533 (1998).</li><li>Kakazu, N., 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=Development+of+spectral+colour+banding+in+cytogenetic+analysis.">Development of spectral colour banding in cytogenetic analysis.</a> Lancet. 357, 529-530 (2001).</li><li>Rithidech, K. N., Honikel, L., Whorton, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mFISH+analysis+of+chromosomal+damage+in+bone+marrow+cells+collected+from+CBA/CaJ+mice+following+whole+body+exposure+to+heavy+ions+(56Fe+ions).">mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (56Fe ions).</a> Radiat Environ Biophys. 46, 137-145 (2007).</li><li>Abrahams, B. S., 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=Metaphase+FISHing+of+transgenic+mice+recommended:+FISH+and+SKY+define+BAC-mediated+balanced+translocation.">Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation.</a> Genesis. 36, 134-141 (2003).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+scoring+of+FISH-&amp;#34;painted&amp;#34;+chromosome-type+exchange+aberrations.">On the scoring of FISH-&amp;#34;painted&amp;#34; chromosome-type exchange aberrations.</a> Mutat. Res. 307, 345-353 (1994).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+"painting"+patterns+resulting+from+complex+exchanges.">FISH "painting" patterns resulting from complex exchanges.</a> Mutat. Res. 312, 51-60 (1994).</li><li>Tucker, J. D., 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=PAINT:+a+proposed+nomenclature+for+structural+aberrations+detected+by+whole+chromosome+painting.">PAINT: a proposed nomenclature for structural aberrations detected by whole chromosome painting.</a> Mutat. Res. 347, 21-24 (1995).</li><li>Knehr, S., Zitzelsberger, H., Bauchinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH-based+analysis+of+radiation-induced+chromosomal+aberrations+using+different+nomenclature+systems.">FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems.</a> Int J Radiat Biol. 73, 135-141 (1998).</li><li>Lucas, J. N., 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=Rapid+translocation+frequency+analysis+in+humans+decades+after+exposure+to+ionizing+radiation.">Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation.</a> Int J Radiat Biol. 62, 53-63 (1992).</li><li>Braselmann, H., 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=SKY+and+FISH+analysis+of+radiation-induced+chromosome+aberrations:+a+comparison+of+whole+and+partial+genome+analysis.">SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.</a> Mutat. Res. 578, 124-133 (2005).</li><li>Sokolova, I. A., 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=The+development+of+a+multitarget,+multicolor+fluorescence+in+situ+hybridization+assay+for+the+detection+of+urothelial+carcinoma+in+urine.">The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.</a> J Mol Diagn. 2, 116-123 (2000).</li><li>Lindholm, C., 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=Biodosimetry+after+accidental+radiation+exposure+by+conventional+chromosome+analysis+and+FISH.">Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH.</a> Int J Radiat Biol. 70, 647-656 (1996).</li></ol>

The authors declare that they have no competing financial interests.

몇 가지 중요한 단계는 mFISH과 SKY의 성공을 결정합니다. 첫번째 가장 중요한 단계는 골수 단핵 세포의 생체 유사 분열 정지를위한 콜히친 처리를 최적화한다. 콜히친 농도와 처리 시간을 개별적으로 또는 콘서트에 효과적인 염색체 그림의 유사 분열 지수뿐만 아니라 염색체 응축 - 두 가지 중요한 전제 조건을 결정한다. 높은 콜히친 농도 이상 처리 시간은 적절한 변성과 하이브리드에 대한...

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations1, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions2. Studies by various groups have shown that stable aberrations are associated with the pathogenesis and development of a number of diseases, including cancer3. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation4. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry5. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements. 
In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes6. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.
Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic ("cryptic") rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.
The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)11-14, radiation carcinogenesis risk assessment15, as well as detection and risk estimation of radiotherapy-related secondary cancer16. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure17. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

<table><tbody><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>221198-100ML</td><td></td></tr><tr><td>SSC Buffer 20&amp;times; Concentrate</td><td>Sigma-Aldrich</td><td>S6639-1L</td><td></td></tr><tr><td>SKY Laboratory Reagent for Mouse</td><td>Applied Spectral Imaging</td><td>FPRPR0030/M40</td><td></td></tr><tr><td>CAD - Concentrated Antibody Detection Kit</td><td>Applied Spectral Imaging</td><td>FPRPR0033</td><td></td></tr><tr><td>Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua</td><td>Applied Spectral Imaging</td><td>FPRPR0182/10</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>12-545B</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>Hydrochloric acid, 37%, Acros Organics</td><td>Fisher Scientific</td><td>AC45055-0025&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Glass Staining Dishes&amp;nbsp; with Screw Cap</td><td>Fisher Scientific</td><td>08-816</td><td></td></tr><tr><td>KaryoMAX Potassium Chloride Solution&amp;nbsp;</td><td>Life Technologies</td><td>10575-090</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Colcemid powder</td><td>Fisher Scientific</td><td>50-464-757&amp;nbsp;</td><td></td></tr><tr><td>Histopaque-1083&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10831</td><td></td></tr><tr><td>Shepherd Mark I, model 25&lt;sup&gt; 137&lt;/sup&gt;Cs irradiator</td><td>J. L. Shepherd &amp;amp; Associates</td><td>Model 484B</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD Biosciences</td><td>647911</td><td></td></tr><tr><td>Ethyl Alcohol, 200 Proof</td><td>Fisher Scientific</td><td>MEX02761</td><td></td></tr><tr><td>PBS, (1x PBS Liq.), w/o Calcium and Magnesium</td><td>Fisher Scientific</td><td>ICN1860454</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10-437-010</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454SK-4</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>AC295320010</td><td></td></tr><tr><td>Zeiss Microscope</td><td>Zeiss</td><td>AXIO Imager.Z2</td><td></td></tr></tbody></table>

detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

전신 방사선 조사 된 마우스의 골수 세포에서 다수의 염색체 수차를 유도한다. 현재 프로토콜은 방사선 조사 후 골수 세포의 생체 유사 분열 정지를 위해 최적화되고, 밀도 구배 원심 분리, 중기 세포 퍼짐의 제조 및 후속 의해 조사 생쥐 골수 단핵 세포의 분리의 뒷다리 골수 세포의 수확 mFISH과 SKY 기술에 의해 방사선 유발 안정적인 염색체 이상 검출. 염색체 이들 ...

Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

여러 형광에 의한 간 염색체 안정 수차의 검출<em&gt; 현장에서</em&gt; 하이브리드 (mFISH) 및 방사선 조사 마우스의 스펙트럼 핵형 분석 (SKY)

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

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Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

9.8K 조회수.  University of Arkansas for Medical Sciences. The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation. 

비디오: Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

종양의 전이에 저산소증의 테스트 효과에 대한 생체 내 모델에서

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

연구

암 연구학

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

단일의 검사 및 포유류 세포에서 이중 가닥 브레이크 수리에 대 한 레이저 마이크로-방사선의 응용

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polycystic 신장 질환과 함께 인간과 마우스의 염색체 이상을 공부하는 스펙트럼 Karyotyping

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

의학

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

인간 결장 암 세포에 있는 DNA 손상 및 복구 Foci의 면역 형광 화상 진찰

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

인간 말초 혈액 림프구의 γ-H2AX 포시 분석에 대한 자동화된 현미경 채점 방법

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

면역학 및 감염병학

마우스의 Prophase-Arrested 난모세포에서 DNA 손상 확인

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

저자 스포트라이트: 포유류 난모세포와 착상 전 배아의 DNA 손상 반응 이해 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

마우스 난모세포에서 DNA 이중 가닥 절단 검출

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

발생학

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics : 수치과 구조 변경의 탐지는 모두 24 인간의 염색체로 동시에 소설 OctoChrome 생선 분석을 사용

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

여러 형광에 의한 간 염색체 안정 수차의 검출

요약

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종양의 전이에 저산소증의 테스트 효과에 대한 생체 내 모델에서

단일의 검사 및 포유류 세포에서 이중 가닥 브레이크 수리에 대 한 레이저 마이크로-방사선의 응용

Polycystic 신장 질환과 함께 인간과 마우스의 염색체 이상을 공부하는 스펙트럼 Karyotyping

인간 결장 암 세포에 있는 DNA 손상 및 복구 Foci의 면역 형광 화상 진찰

인간 말초 혈액 림프구의 γ-H2AX 포시 분석에 대한 자동화된 현미경 채점 방법

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

마우스 난모세포에서 DNA 이중 가닥 절단 검출

마우스 난모세포에서 DNA 이중 가닥 절단 검출

마우스 난모세포에서 DNA 이중 가닥 절단 검출

Chromosomics : 수치과 구조 변경의 탐지는 모두 24 인간의 염색체로 동시에 소설 OctoChrome 생선 분석을 사용