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 ...

chromosomics-detection-numerical-structural-alterations-all-24-human

Research

JoVE Journal

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

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

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

리그노셀룰로오스성 자료에 대한 높은 처리량 당화 분석

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

자동을 사용하여 염색체의 높은 처리량 물리적 매핑<em&gt; 현장에서</em&gt; 하이브리드화

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

엑손 캡처 및 대규모 병렬 염기 서열 종양 검체에서 체세포 유전 변경을 검출

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

메추라기 오리 키메라를 사용하여 두개 안면 개발에 종 - 특정 포스트 평가

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

소설 및 액세스 접근 : 구조 HEK 293F 서스펜션 세포의 생물학에 대한 재조합 단백질 발현

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

형광 표지 된 나노 입자를 사용하여 쥐, 인간과 돼지 유래의 살아있는 세포에서 세포 내 유전자 발현의 검출

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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

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