저자들은 비판적으로 읽고 원고를 편집 박사 Cliona M. 맥헤일을 감사드립니다. 이 작품은 환경 보건 과학, 패 장 건강과 기금 R01ES017452 국립 연구소의 국립 연구소에 의해 재정 지원되었다.

1. 샘플 슬라이드 준비 <ul><li> OctoChrome 생선을 포함하여 인간 세포의 확산 metaphase의 염색체 변화를 검토하도록 설계하지만 교양 말초 혈액 세포, 줄기 / 전구 세포 및 세포 라인에 국한되지 않습니다. 샘플은 metaphases에게 3,11-13 수확을위한 표준 절차에 따라 작성하여야한다 : 세포를 팽창하는 metaphase, hypotonic 치료 세포를 체포하고 colcemid 치료를 채용하여, 약 0.5-1 X 10 6 세포에서 정학에 세포를 해결하는 Carnoy의 정착액 ML 당. </li><li> 2 분 동안 순수한 에탄올 파버가 : 8 사각형 슬라이드 (PMP 803, Cytocell 공사, 캠브리지, 영국, 그림 2 OctoChrome 키트, 카탈로그 #에서 제공) 청소합니다. </li><li> 샘플 슬라이드를 만들기 전에, 세포 밀도를 확인하기 위해 슬라이드에 세포를 소량 놓습니다. 세포 밀도는 (너무 많이 중복되는 세포) 너무 높은 경우 신선한 정착액으로 현탁액을 희석. 세포 밀도는 (역시 몇 가지 세포 너무 낮으면슬라이드들), 세포를 다운 스핀과 신선한 정착액의 작은 볼륨에서 다시 일시 중지합니다. 8분의 2 - - 5분의 3 - 육분의 사 사각형 7분의 1을 번갈아 일련의 템플릿 슬라이드의 8 각 영역 진입 세포 현탁액의 피펫 5-10 μl. 처음 두 사각형 공기 건조되면 동일한 방식으로 나머지 사각형을 더럽힐. 이것은 서로 방해로부터 세포 확산을 방지할 수 있습니다. </li><li> 마른 슬라이드 하이브리드화 이전보다 5 년 동안 질소 분위기에서 -20 ° C에 저장할 수 있습니다. </li></ul> 2. 자동화 시스템을 사용하여 metaphases의 현지화 <ul><li> 슬라이드의 각 절반 센터는 4의 20 μl &#39;, 6 diamidino-2-phenylindole (0.2 μg / ML)을 적용하고 coverslip으로 덮어. </li><li> Metafer 소프트웨어 (MetaSystems, Altlussheim, 독일)를 사용하여 metaphase 세포를 집중하는 샘플 슬라이드의 자동 스캔을 수행합니다. 이것은 모든 metaphases와 미래의 모든 세포 이미지의 재평가의 위치를​​ 용이하게합니다. </li><li> 수동으로 검사 결과를 확인하고 비 metaphase 유물을 삭제합니다. </li></ul> 3. 이종 교잡 <ol><li> 샘플 슬라이드와 OctoChrome 장치의 준비 </li><ul><li> DAPI 멀리 씻어 30 분 동안 실온에서 2X SSC 버퍼의 샘플 슬라이드를 찍어. </li><li> 에탄올 세차장 (2 70% 분만에 각각 85 % 및 100 % 에탄올)의 시리즈를 통해 샘플 슬라이드를 탈수, 건조하고 37 시간을 15 분 ° C 열판에 게재할 수 있습니다. </li><li> 37 ° C의 물을 욕조에 Chromoprobe의 Multiprobe의 하이브 리다이 제이션 회의소를 (OctoChrome 키트에 제공) 장소 37에 평형 수 있도록 ° C (+ / - 1 ° C). </li><li> 37 ° C.에 OctoChrome 장치마다 반복 pipetting 미리 따뜻한 25 μl 나누어지는하여 하이브 리다이 제이션 솔루션 (OctoChrome 키트에서 제공) 믹스 </li><li> 미리 따뜻하게 각 OctoChrome 장치 ° C 열판에 장치 라벨 면을 아래로 배치하여 37로 (OctoChrome 키트에 제공된 그림 2 참조). T를 만지지 마십시오그는 OctoChrome 장치의 양각 표면들은 프로브를 포함하고 있습니다. </li></ul><li> OctoChrome 장치를 통해 샘플 슬라이드의 위치 (그림 2) </li><ul><li> 그것이 37로 유지하면서 미리 예열 OctoChrome 장치의 8 각 영역에 미리 예열 하이브 리다이 제이션 용액 2 μl를 추가 ° C. </li><li> 지금은 거꾸로입니다 숫자 1, OctoChrome 장치의 오른쪽 상단 핸드 영역에 위치해있는 등 OctoChrome 장치로 템플릿 슬라이드를 조심스럽게로 바꾸란. 정사각형 1을 찾을 수 있도록 장치의 위치는 오렌지에 표시되었습니다. </li><li> 템플릿 슬라이드 신중 OctoChrome 장치에 일치하는 영역으로 정렬되어 있는지 확인합니다. 하이브 리다이 제이션 솔루션의 드랍스는 슬라이드와 접촉하도록 조심스럽게 OctoChrome 장치를 통해 슬라이드를 낮춥니다. 부드러운 적용, 심지어 하이브 리다이 제이션 솔루션 OctoChrome 장치에 제기된 각 영역의 가장자리에 확산되도록해야할 듯. </li><li> 들어올려슬라이드 / OctoChrome은 신중하게 유리 슬라이드의 서리낀 끝을 잡고, 그리고 슬라이드 OctoChrome 장치 아래가되도록 바꾸란. 이 프로브의 교차 오염을 일으킬 수로 장치 템플릿 슬라이드를 통해 비방하지 않도록 확인하십시오. </li><li> 37 장 소 ° C (+ / - 1 ° C)에 10 분 열판. </li></ul><li> 변성 </li><ul><li> 그 수준을 보유하는 특정 돌보는 열판에 샘플 슬라이드 / OctoChrome 장치를 전송합니다. 샘플 슬라이드 균일 연락처 열판을 보장합니다. </li><li> 75시 열판에 변성 ° C (+ / - 1 ° C) 5 분. </li></ul><li> 이종 교잡 </li><ul><li> Chromoprobe의 Multiprobe의 하이브 리다이 제이션 상공 회의소에서 개최 샘플 슬라이드 / OctoChrome 장치. </li><li> 뚜껑을 교체하고 37에서 챔버는 역부족 ° C (+ / - 1 ° C) 물 목욕 (비 - 저어) 하룻밤 사이에. 참고 : 하이브리드화 챔버에 뚜껑을 밀봉하지 마십시오; 물을 욕조에 뚜껑을 밀봉하지 마십시오;에 잡종을하지 마십시오보육. 이 단계에서는 습도 조절에 중요​​합니다. </li></ul><li> 포스트 하이브 리다이 제이션 세차장 </li><ul><li> 세차 솔루션의 준비 해결 방법 1 : 0.4x SSC를 포함 Coplin / Hellendahl 항아리를 준비합니다. 72로 평형 ° C (+ / - 1 ° C) 물 목욕과 7.0으로 산도를 조정합니다. 해결 방법 2 : 2X SSC와 0.05 % 십대 초반 20를 포함하는 Coplin / Hellendahl 항아리를 준비합니다. 실온에서 서서하도록 허용합니다. </li><li> 슬라이드에서 조심스럽게 OctoChrome 장치를 제거하고 2 분 동안 해결 방법 1에서 슬라이드를 넣습니다. </li><li> 30 초 동안 솔루션이로 슬라이드를 놓습니다. </li></ul></ol> 4. 장착 및 결과의 시각화 <ul><li> 슬라이드의 각 절반 센터에 DAPI 20 μl를 적용하고 coverslip을 적용합니다. </li><li> 형광 현미경으로 보는 전에 실온에서 10 분 동안 어둠 속에 품어. </li></ul> 5. 대표 결과 Figu다시 3A는 스퀘어 2에서 정상 metaphase 세포를 보여줍니다. (1) - (3) : 염색체 8, 21, 12이 적색, 녹색, 파란색 페인트, 각각 텍사스 빨강, FITC, 그리고 딕 필터를 통해 시각입니다되었다 (4) : DAPI / FITC /를 통해 시각화 텍사스 레드 삼중 필터. 일반적으로, 블루와 그린 염색체는 삼중 필터를 통해 명확하지 있으며 특정 딕 필터 아래에서 볼해야합니다. 그림 3B 백혈병 특정 염색체 translocation과 aneuploidy와 대표 비정상 세포를 보여줍니다. (1) : T (8, 21), 급성 골수양 백혈병에서 흔한 염색체 translocation, (2) : Trisomy 21, 염색체 21, 백혈병의 일반적인 aneuploidy 세 복사합니다. <img alt="그림 1" src="/files/ftp_upload/3619/3619fig1.jpg" /> 그림 1. OctoChrome 장치와 예상되는 염색체 페인팅 결과에서 염색체 조합의 정렬. <img alt="그림 2" src= &quot;/ files/ftp_upload/3619/3619fig2.jpg&quot;/&gt; 그림 2. OctoChrome 장치를 통해 샘플 슬라이드의 위치. 그림 3. 대표 결과OctoChrome 물고기 (스퀘어 2)에서 얻은. <img alt="그림 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> 그림 3A. 광장 2에 그린 염색체 8, 12, 21와 정상 세포. <img alt="그림 3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> 그림 3B. 광장 2 백혈병 특정 염색체 translocation과 aneuploidy와 대표 비정상 세포.

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우리는 공개 할게 없다.

이 소설 OctoChrome - 물고기 분석은 하나가 동시에 슬라이드에 하나의 하이브리드화 모두 24 인간의 염색체를 검사 할 수 있습니다. 8 사각형의 염색체 조합의 배치는 가장 일반적인 leukemias과 lymphomas의 비 임의 염색체 rearrangements의 식별을 용이하게하도록 설계되었습니다. 따라서 분석이 동시에 숫자 (aneuploidy)뿐만 아니라 구조적인 (translocations) 염색체 변경 감지할 수 있습니다. ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td><tr><tr><td> OctoChrome 키트 </td><td> Cytocell (주), 캠브리지, 영국 </td><td> PMP 803 </td><td></td></tr><tr><td> phytohaemagglutinin (PHA) </td><td> Invitrogen 주식 회사 </td><td> 10576-015 </td><td></td></tr><tr><td> Colcemid </td><td> Invitrogen 주식 회사 </td><td> 15212-012 </td><td></td></tr><tr><td> Carnoy의 정착액 </td><td></td><td></td><td> 메탄올 : 빙초산 = 3 : 1 </td></tr><tr><td> 형광 마일croscope </td><td></td><td></td><td> 동시에 서로 다른 색상을 볼 수 DAPI, 텍사스 빨강, FITC와 Coumarin의 스펙트럼 개별적 및 DAPI / FITC / 텍사스 레드 삼중 필터를 볼 수있는 필터가 장착된 </td></tr><tr><td> Metafer 소프트웨어 </td><td> MetaSystems, Altlussheim, 독일 </td><td></td><td> 모든 metaphases를 찾습니다 촉진하고 이상에게 재평가 </td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

원위치 하이브리드화 (물고기)의 형광은 특정 DNA 시퀀스는 세포 핵 1 metaphase이나 계면 염색체를 감지할 수 있도록하는 기법이다. 기술은 전체 염색체 또는 특정 염색체 지역에 잡종을 고유한 시퀀스와 DNA 프로브를 사용하며 고전 cytogenetics에 대한 강력한 보조 역할을합니다. 예를 들어, 많은 이전의 연구는 고전 cytogenetic 분석 2를 사용하여 벤젠 노출, 벤젠 - 중독 환자 및 벤젠에 노출된 건강한 노동자와 관련된 백혈병 환자의 증가 염색체 aberrations의 자주 감지했다. 물고기를 사용하여, 백혈병 특정 염색체 변경은 벤젠 유발 leukemogenesis의 cytogentic 변화의 중요한 역할을 나타내는 벤젠 3-6에 노출 분명히 건강한 노동자로 상승하는 관찰되었습니다. 일반적으로, 하나의 생선 분석은 조사 하나 또는 몇 전체 염색체또는 슬라이드마다 특정 loci, 그래서 여러 hybridizations는 인간의 염색체를 모두 충당하기 위해 여러 슬라이드에 실시해야합니다. 분광 karyotyping (SKY)는 동시에 전체 게놈의 시각화 있지만, 특별한 소프트웨어 및 장비 제한 그 응용 7에 대한 요구 사항을 허용합니다. 여기서는 소설 생선 분석, 우리는 그러한 염색체 전체 aneuploidy 연구와 같은 인간의 연구에서 한 슬라이드에있는 모든 24 인간의 염색체의 동시 분석 (CWAS)로 여기에 정의 Chromosomics에 대해 적용할 수있는 OctoChrome - 물고기, 설명 8. Chromoprobe의 Multiprobe 시스템으로 Cytocell하여 판매 방법의 기초는 세 가지 모든 염색체 페인팅 프로브 (그림 1) 운반 각각 8 사각형으로 나뉘어져 있습니다 OctoChrome 장치입니다. 세 프로브 각각 직접 가지 다른 색의 형광단, 녹색 (FITC), 빨간색 (텍사스 빨강), 파랑 (Coumarin)로 표시됩니다. OctoChrom의 염색체 조합의 정리, T (15, 17), T (8, 21, 전자 장치 인스턴스 t (22 9)에 대해 가장 일반적인 leukemias과 lymphomas에서 발견되지 않은 임의의 구조적 염색체 변경 (translocations)의 식별을 용이하게하도록 설계되었습니다 ), T (14, 18) 9. 또, 염색체의 숫자 변화 (aneuploidy)가 동시에 감지가 가능하다. 해당 템플릿 슬라이드도 metaphase의 확산은 (그림 2) 구속되며, OctoChrome 장치로 배치되는 방향 8 사각형으로 나뉘어져 있습니다. 프로브 및 대상 DNA는 높은 온도와 습기 챔버에 hybridized에 변성이며, 다음 모든 24 인간의 염색체는 동시에 시각이 될 수 있습니다. OctoChrome 물고기는 백혈병과 림프종의 임상 진단 및 모든 염색체의 aneuploidies의 검출을위한 유망 기술입니다. 우리는 벤젠 노출 중국어 노동자 8,10의 CWAS 연구에서이 새로운 Chromosomic 접근 방식을 적용했습니다.

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

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

소설의 형광<em&gt; 현장에서</em&gt; 하이브리드화 (물고기) 동시에 특히 숫자 및 구조 모두 염색체 변경 한 하이브리드화에서 하나의 디바이스에있는 모든 24 인간의 염색체의 백혈병 및 림프종과 관련된 특정 염색체 translocations를 검사 방법은 설명되어 있습니다.

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 ...

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Biology

18.8K 조회수.  University of California, Berkeley. 소설의 형광 현장에서 하이브리드화 (물고기) 동시에 특히 숫자 및 구조 모두 염색체 변경 한 하이브리드화에서 하나의 디바이스에있는 모든 24 인간의 염색체의 백혈병 및 림프종과 관련된 특정 염색체 translocations를 검사 방법은 설명되어 있습니다.

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

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.

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 ...

연구

유전학

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

사전 이식 유전자 진단 생선

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

의학

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

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 ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

비 형질 인간 섬유 아세포의 증식 배체 세포의 수립

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

발생학

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

유사 분열 동안 염색체 분리의 라이브 셀 이미징

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Fluorescence In Situ Hybridization을 사용한 난소 세포의 X 염색체 이상 탐색

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

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

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

마우스 난 모세포의 스핀들 어셈블리 체크 포인트 무결성 평가

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...