We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. 염색체 준비 <ol><li> 종자 발아 및 뿌리 끝의 고정 <ol><li> 22 ~ 24 ℃에서 2 일간 어두운 조건에서 배양 접시에 촉촉한 필터 종이의 두 층에 10 ~ 20 보리 씨앗을 발아. 면도날을 사용하여 시드로부터 1-2cm의 길이 격렬한 뿌리 잘라. </li><li> 분쇄 된 얼음 - 물에 차가운 수돗물을 함유하는 500 ㎖의 유리 병을 배치하여 빙 냉수를 준비한다. 빙 냉수를 폭기 및 중기 세포의 빈도를 증가시키기 위해 20 시간 동안 뿌리 끝 부분을 넣는다. </li><li> 50ml의 에탄올에 물을 뿌리 전송 : 아세트산 (3 : 1)을 고정 제 2 일 동안 실온에서이를 해결하기 위해. 갓 준비 에탄올에 보관 뿌리 : 아세트산 (3 : 1) 4 ° C에서 정착 년까지 사용할까지. </li></ol></li><li> 세탁 효소 처리 <ol><li> 5 분 50 회 mL 유리 비커를 사용하여 30 mL의 얼음처럼 차가운 수돗물로 씻어 뿌리 10-20. 쌍안 현미경을 사용합니다. 에 의해 뿌리 하나를 전송30 mL의 0.01 M 시트 레이트 완충액 (+ 0.01 M 시트르산 나트륨, 0.01 M 시트르산, pH를 4.8) 겸자를 사용하여 5 분 동안 두번 유리 비커를 흔들어 씻는다. 완전히 액체를 제거하고 차단 원하지 않는 비 분열 조직을 면도날을 사용하여 필터 종이에 뿌리를 놓습니다. </li><li> 시계 접시로 식물 조직을 부드럽게 약 50 분 (표 1), 37 ° C에서 1 ㎖의 효소 혼합물에 20 뿌리 끝까지 배양한다. 효소 혼합물 0.01 M 구연산 버퍼에 희석 0.7 % 셀룰라아제 R10, 0.7 %의 셀룰라아제, 1 % pectolyase과 1 % cytohelicase가 포함되어 있습니다. -20 ℃에서 효소 혼합물을 저장하고 다섯 번까지 재사용. </li><li> 피펫으로 효소를 제거하고 잔류 효소를 대체하기 위해 두 번 5 ml의 0.01 M 구연산 버퍼와 얼음에 뿌리 끝을 씻는다. </li></ol></li><li> 루트 침용 <ol><li> 두 번주의 깊게 같은 시계 접시에 1 ml의 96 % 에탄올로 세척 루트 팁. 갓 준비 고정액 (25 % 에탄올 75 % 아세트산)와 에탄올을 교체합니다. 10 ~ 15를 사용하여루트 팁 당 μL 정착. </li><li> 해부 바늘이나 집게로 2 ML 튜브에 정착와 함께 전송 루트 팁과 분해 루트 분열 조직. 세포 현탁액을 얻기 위해 세포를 다시 중단 튜브 20 번 눌러. 2 개월 -20 ° C 최대의 세포 현탁액을 저장합니다. </li></ol></li><li> 세포 현탁액의 적하 <ol><li> 50 ° C에서 뜨거운 접시에 물을 적신 종이 조직의 2 ~ 3 층을 놓습니다. 30 분 동안 냉장고에 얼음처럼 차가운 수돗물에 현미경 슬라이드를 담가. 장소는 축축한 종이 티슈 위에 슬라이드. </li><li> 피펫 7-10 세포 현탁액의 μL와 핫 플레이트에 배치 된 냉각 슬라이드에 20cm의 거리에서 드롭합니다. 피펫 (10) 슬라이드에 세포 현탁액과 같은 장소에 아세트산 - 에탄올 혼합물의 μL 및 추가 2 분 동안 핫 플레이트에 슬라이드를 유지. 물티슈없이 핫 플레이트에 슬라이드를 배치하고 1 분 동안 건조 할 수 있습니다. </li></ol></li><li> 품질 관리 및 스토리지 박슬라이드의 전자 <ol><li> 염색체 스프레드의 품질을 제어하는​​ 위상차 현미경을 사용하여 슬라이드를 확인한다. -20 ° C에서 코 플린 항아리에 96 %의 에탄올에 침지하여 같은 날 또는 저장 중 슬라이드를 사용합니다. </li></ol></li><li> 물고기 전에 슬라이드 전처리; 모든 단계는 실온에서 수행 <ol><li> 배 SSC의 50 ㎖를 포함하는 코 플린 항아리에 넣어 슬라이드 5 분 (20 배 SSC는 3 M의 NaCl 300 MM의 트리 소듐 시트 레이트를 포함). 3-10 분 동안 45 % 아세트산 50 ㎖를 함유하는 코 플린 항아리 집게, 이전 슬라이드를 사용. </li><li> 전달은 10 분 동안 2 배 SSC 50 ㎖를 함유하는 코 플린 항아리에 슬라이드. 10 분 동안 (2 × SSC)에 4 % 포름 알데히드의 50 ㎖를 포함하는 코 플린 항아리로 전송 슬라이드 빠져들게 슬라이드 염색체를 해결하려면. </li><li> 50 ml의 2 배 SSC를 포함하는 코 플린 항아리, 4 분 각각의 슬라이드 3 회 세척하여 포름 알데히드를 제거합니다. 수직에서 70 %의 시리즈에서 2 분, 90 %, 100 % 에탄올, 각각 건조 슬라이드의 코 플린 항아리에 슬라이드를 탈수위치. </li></ol></li></ol> 제자리 하이브리드 2. 형광 (물고기) <ol><li> 각 슬라이드에 대해, 탈 포름 아미드 10 μL, 4X 혼성화 완충액 5 μL를 사용하여 총 20 μL의 혼성화 용액을 제조 하였다 (200 ㎕를 완충액 80 ㎕의 20 배 SSC, 8 ㎕의 1 M 트리스 -HCl pH가 8.0를 포함 1.6 ㎕의 0.5 M EDTA, 11.2 μL 10 μg의 / μL 연어 정자와 99.2 μL의 DNase를없는 물), 프로브의 3 μL와 DNase를없는 물 2 μL. </li><li> 24 X 32mm 커버 슬립과 슬라이드 커버 당 혼성화 용액 20 μl를 추가하고 고무 시멘트와 커버 슬립을 체포. 핫 플레이트에서 2 분 동안 80 ℃에서 프로브와 동시에 슬라이드 변성. </li><li> 전송 습기 챔버에 슬라이드와 37 ° CO / N 피 빛 슬라이드를 품어. 배 SSC와 코 플린 항아리에 슬라이드를 세척하여 커버 전표를 제거합니다. 그리고 55-60 ° C의 2 배 SSC를 포함하는 코 플린 항아리에 넣어 슬라이드 20 분 동안 품어. </li><li> 실온에서 2 분 동안 코 플린 항아리에 SSC를 2 배 슬라이드를 놓습니다. 각각 70 %, 90 %, 100 % 에탄올, 일련의 2 분 동안 코 플린 항아리 슬라이드 탈수. </li><li> 강렬한 빛을 방지, antifade 설치 매체에 / ㎖ 4 &#39;, 6 diamidino-2-phenylindoline 1 μg의 (DAPI)과 슬라이드와 Counterstain과를 공기 - 건조. </li></ol> 3. 현미경 분석 및 저장 <ol><li> 표면 형광 현미경을 사용하여 슬라이드를 분석한다. 필터의 선택은 라벨 프로브 형광 색소 사용에 의존한다. 필요한 경우, 1 년까지 어두운 조건에서 4 ° C에서 저장 슬라이드. </li></ol>

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The authors have nothing to disclose.

염색체 준비 실험 잔디 가족 (벼과)에 속하는 시리얼 어린 뿌리를 이용하여 수행되었다. 모든 분석 종은 이배체 게놈 세트 14 상대적으로 긴 유사 분열 중기 염색체 (11 ~ 15 μm의)가 큰 게놈 종 (5.1-7.9 GBP)에 속한다. 발아 한 뿌리의 길이가 세포 분열 조직의 최대치를 얻기 위해 2 cm 이상이 아니었다. 분열하는 세포의 동기화 유사 분열 중기의 수량이 10 퍼 향상 20 시간 긴 ...

계내 혼성화 (FISH)의 형광은 염색체 수준에서 단일 및 높은 복사 서열의 물리적 맵핑에 대한 효과적인 도구이다. 전제 조건은 높은 품질의 염색체 스프레드의 준비입니다. 동물과 식물 세포에 대한 동등하게 적합 할 것이 더 일반적인 염색체 준비 프로토콜이 없습니다. 식물 염색체의 준비로 인해 다른 종 내에서 단단한 세포벽과 각종 세포질의 일관성에 특히 도전이다. 식물 염색체의 제조를위한 바람직한 방법 중 하나는 드롭 확산 기법 및 공기 - 건조 기술로 알려진 1,2 소위 드롭 기술이다. 이 방법은 처음 체외 성장 포유 동물 세포 3 Rothfels과 Siminovitch에 의해 1958 년에 도입되었다. 나중에 마틴 등 알. 4 카토 등. 5는 식물이 방법을 적용. 최근 방법이라는 &#39;SteamDrop &amp;# 39; 비 중첩 염색체 (6)의 제조에 수증기를 사용하는 개발되었다. 높은 습도의 긍정적 인 영향은 이전 관찰되었지만 7 &#39;SteamDrop&#39;는 고품질 염색체 제제 (6)의 제어 흐름을 제공한다. 증기 처리는 아마도 염색체 단백질의 일부​​ 수정에 접속 염색체 연신시킨다. 전체 중기의 충분한 수의 고정 후속 물고기 실험은 기술적 인 전문 지식을 필요로 확산 있지만 중기 스프레드 결과의 품질은 매우 높다. 여기에서 우리는 단일 및 높은 사본 프로브 5,8의 물고기 검출에 적합한 유사 분열 시리얼 염색체의 제조를위한 프로토콜을 제시한다. 이 방법은 카토 50 % -55 %의 상대 습도 하에서 수행 (9) (도 1)에 의해 설명 풍건 적하 법의 개선 된 변이체이다. 이 프로토콜은 감소 된 수를 포함그 적용이 쉽고 효율적이고 재현하게 세척 단계. 우리가 Hordeum vulgare의, 수평 bulbosum, 수평 marinum, 수평 murinum, 수평 pubiflorum 및 Secale의 cereale 높은 품질의 염색체 확산과 물고기 결과를 얻었다이 프로토콜을 사용.

<table>
	<tbody>
		<tr>
			<td>Hot Plate</td>
			<td>MEDAX GmbH</td>
			<td>12603</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase R10</td>
			<td>Duchefa</td>
			<td>C8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase&#160;</td>
			<td>CalBioChem</td>
			<td>219466</td>
			<td></td>
		</tr>
		<tr>
			<td>Pectolyase</td>
			<td>Sigma</td>
			<td>P3026</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytohelicase</td>
			<td>Sigma</td>
			<td>C8274</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-12-dUTP</td>
			<td>Invitrogen</td>
			<td>C3176</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluor488-5-dUTP</td>
			<td>Invitrogen</td>
			<td>C11397</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluorecsence microscope</td>
			<td>Olympus BX61</td>
			<td>BX61</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Orca ER, Hamamatsu</td>
			<td>C10600</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>fluorecsent dye</td>
		</tr>
	</tbody>
</table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

염색체 스프레드의 제조 현장 하이브리드에 형광의 성공적인 성능 (FISH)을위한 전제 조건이다. 높은 품질의 식물 염색체 스프레드의 준비로 인해 단단한 세포벽에 도전한다. 식물 염색체의 제조를위한 승인 방법 중 하나는 소위 드롭 제제, 또한 드롭 확산 또는 공기 - 건조 기술로 알려져있다. 여기, 우리는 유사 분열 염색체의 빠른 준비를위한 프로토콜은 단일 및 높은 사본 DNA 프로브의 물고기 검출에 적합한 확산 제시한다. 이 방법은 50 % -55 %의 상대 습도 하에서 수행 풍건 드롭 방식의 개선 된 변이체이다. 이 프로토콜은 그 적용이 쉽고 효율적이고 재현하게 세척 단계의 감소 된 수를 포함한다. 이 방법의 명백한 이점은 성공적인 FISH 분석을위한 완벽한 전제 조건으로서 잘 확산, 손상되지 않은 수많은 중기 염색체이다. 이 프로토콜을 이용하여, 우리는 높은 품질을 얻을 CHROMosome 스프레드와 Hordeum vulgare의, 수평 bulbosum, 수평 marinum, 수평 murinum, 수평 pubiflorum 및 Secale의 cereale에 대한 재현 물고기 결과.

유사 분열 중기 스프레드 현미경 슬라이드는 전술 빠른 공기 건조 적하 염색체의 제조 방법에 의해 제조 하였다 (SUPPL.도 1). FISH 분석은 모두, 반복적이고 단일 - 카피 시퀀스를 사용하여 수행 하였다. 이미지는 대응하는 형광체의 여기를 가능 필터 세트 표면 형광 현미경에 의해 얻은 고감도 CCD 흑백 카메라에 의하여 촬영되었다. 화상 획득을 위해 우리는 화상 취득 소프트웨어가 설치...

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

식물에서 물고기에 적합한 빠른 공기 건조 떨어 뜨리 염색체 제조 방법

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

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13.1K 조회수.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

비디오: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : 정확한 Genotyping을위한 간단한 방법

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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생물학

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

항체 라벨링에 대한 Drosophila Polytene의 염색체 Squashes의 준비

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

얼음 총액 : 성장을위한 방법 Arabidopsis높은 처리량 Genotyping을위한 96 - 웰 플레이트에와 토마토 식물

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

배양 세포에서 염색체 준비

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

결합 된 DNA-RNA 형광<em&gt; 현장에서</em&gt; 하이브리드 화 (물고기) 차별화 된 여성 마우스 배아 줄기 세포에서 X 염색체의 불 활성화를 연구하는

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

포유류의 전체 배아 문화에 적합 쥐 혈청의 제조

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Meiotic 염색체의 준비에서 마우스 Spermatocytes 확산

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...