우리는 조언과 기술 지원에 대한 Blaschke, Grunwald가, 그리고 비트 버 실험실의 구성원을 감사드립니다. 이 작품은 JLB에 PCMC 재단, NIH R01 MH092256와 DP2 MH100008, 그리고 천 재단 연구 보조금 # 1 - 2013 년 - 425 년 3 월에 의해 지원된다.

1. DNA 준비 <ol><li> 50 mM의 KCl을 10 mM 트리스 - 염산 pH를 8.3, 0.3 % 트윈 20, 0.3 % NP40 : DNA의 용해 버퍼를 준비한다. 사용 12 일에 1 ㎎ / ㎖의 최종 농도로 신선한 테 K 추가. </li><li> 조직 컬렉션 : <ol><li> 성인 물고기 지느러미 클립 : <ol><li> 물고기를 마취 : 0.004 % MS-222 (tricaine) 솔루션의 물고기를 놓습니다. 아가미의 움직임이 둔화 될 때까지 기다립니다. </li><li> 5-10 킴 와이프의 스택에 물고기를 넣고 멸균 면도날와 꼬리 지느러미, 2 ~ 3 mm의 작은 조각을 잘라. </li><li> 신속하게 복구를 위해 신선한 물을 표시 탱크에 물고기를 배치, 조심스럽게 핀 클립을 포함 할 탱크와 해당 관을 모두 레이블을 붙입니다. 물을 변경하고 매일 물고기를 먹이. </li><li> 멸균 피펫 팁과 핀 클립을 선택하고 100 ㎕의 DNA 용해 버퍼로 채워진 튜브로 전송할 수 있습니다. </li><li> 피펫 팁을 폐기하고, 날카로운 물건 용기에 면도날을 버린다. </li></ol></li><li> 배아의 꼬리 클립 : <ol><li> 0.004 % MS-222 (tricaine) 솔루션으로 배아를 놓습니다. 2 분을 기다립니다. </li><li> 해부 현미경 집게로 꼬리의 조각을 잘라. </li><li> 30 μL의 DNA 용해 버퍼 가득 레이블 튜브에 꼬리를 넣습니다. </li><li> 신속하게 100 ㎕의 4 % 파라 포름 알데히드를 포함하는 튜브에 배아를 넣어. </li><li> 배아 및 해당 꼬리 튜브 라벨 튜브. </li><li> 고정 용 4 ° C에서 하룻밤에 배아와 튜브를 저장합니다. </li><li> 오염을 최소화하기 위해 각 배아 사이에 밀리 Q 물과 킴 와이프를 사용하여 집게를 청소합니다. </li></ol></li><li> 전체 배아의 경우 : <ol><li> 0.004 % MS-222 (tricaine) 솔루션으로 배아를 놓습니다. 2 분을 기다립니다. </li><li> 100 ㎕의 DNA 용해 버퍼로 채워진 튜브에 1-5 배아를 넣어. Dechorionation 필요가 없습니다. </li></ol></li></ol></li><li> DNA 소화 55 ° C에서 조직 (지느러미 또는 꼬리 클립 또는 태아)와 튜브를 품어밤새 12 4 시간 최대를위한. </li><li> 테 K에게 비활성화 15 분 12 ~ 95 ° C의 열 관. DNA는 바로 PCR에 사용, 또는 최대 3 개월 동안 -20 ° C에서 보관해야합니다. </li></ol> 2. PCR <ol><li> 디자인 프라이머 수동 또는 프라이머 설계 소프트웨어 (예 : Lightscanner 뇌관 디자인 소프트웨어 Biofire)에 의해. 르마위한 PCR 제품은 일반적으로 50-200 염기쌍이며 SNP 검출을위한 PCR 제품은 작고, 50-80 염기쌍이어야한다. </li><li> PCR <ol><li> 96도 또는 384 - 웰 플레이트에서 PCR 반응을 수행합니다. </li><li> 10 ㎕의 전체 볼륨을 사용하여 4 μL의 LightScanner 마스터 믹스 (0.1 U 핫 시작의 Taq-AB 중합 효소, 버퍼, 0.2 밀리미터의 dNTPs, 2 mM의 염화 마그네슘 및 1X 형광 이중 가닥 DNA 결합 염료), 5 pmol의 각 프라이머, 성인 꼬리 지느러미 또는 배아의 꼬리 (13)로부터 3 ㎕의 게놈 DNA 추출 1 ㎕의 게놈 DNA 템플릿. </li><li> 30 ㎕의 미네랄 오일을 커버 샘플. </lI&gt; <li> 광학적으로 투명 접착 씰 플레이트를 커버. </li><li> PCR 순환 상태 전형적인 조건은 95 ° C에서 5 분 10 초 30주기를 95 ° C이다 최적화, 60 ° C에서 25 초, 72 ° C에서 30 초, 30 초 동안 95 ° C로 끝나는로 냉각 15 ° C. </li><li> 스토어 PCR의 4 ° C에서 접시 또는 르마로 직접 진행합니다. </li><li> PCR의 초기 최적화 동안 아가 로스 겔 전기 영동을 사용하여 크기를 분석하여 우리의 PCR 생성물을 확인하고, 상기 PCR 생성물의 서열 분석에 의해, 표시하는 경우. </li></ol></li></ol> 3. 르마 <ol><li> 열 플레이트 <ol><li> 용융 분석 시스템에서 접시를 놓습니다. </li><li> (LightScanner 소프트웨어는 전화-IT 2.0) 소프트웨어를 엽니 다. </li><li> 60 ~ 95 ° C.의 열 플레이트 </li><li> 새 데이터 저장소 파일을 만듭니다. </li></ol></li><li> 르마 데이터를 분석 (그림 2). 분석의 여러 단계가 가능합니다. 우리는 데이터 m의 가장 일반적으로 사용되는 세트를 보여anipulations. <ol><li> 부분적인 설정 부분의 탭을 클릭합니다. 샘플 우물을 선택합니다. </li><li> 표준화 <ol><li> 왼쪽 패널의 표준화 탭을 선택합니다. 형광 분산을 제거하려면 수동으로 사전 (초기)의 병렬 더블 라인을 배치하고 후 (최종) - 용융 영역 (그림 2C, 화살촉) 그림과 같이 1과 0으로 모든 샘플의 premelt 및 사후 용융 형광 신호를 정상화 각각 14. </li><li> 용융 영역 전후 멜트 라인 사이의 영역에 있지만, 선택되지 않도록 라인의 위치를​​ 조정한다. 일반적으로, 낮은 최소 및 낮은 최대 (어퍼 최소 어퍼 최대) 온도 선이 떨어져 약 1 ° C를 배치해야합니다. </li><li> 정상화 온도 위치를 선택 모든 샘플 (premelt 지역 및 최소 또는 후 용융 지역 없음 (0 %) 형광의 최대 또는 전체 (100 %)의 형광을 갖도록 POS 최선으로sible). 정규화는 융점과 0의 최소 형광의 융점부터 다음 수평에 가까운 라인까지 연장되어 하나의 형광 극대에서 거의 수평 라인 초래한다 상기 1 또는 0 아래 형광 수준이 없어야 . 예를 들어,도 2c에, 79-80 ° C.의 온도 범위를 사용 premelting 곡선을 정상화 </li></ol></li><li> 그룹화 / 클러스터링 녹는 온도 (그림 2C, 녹색 상자)을 기반으로 유전자형을 구분합니다. 그룹화를 선택합니다. <ol><li> 그룹화 섹션의 표준 선택 목록에서 Autogroup을 선택합니다. </li><li> 그룹화 섹션에서 감도 선택 목록에서 각각 하나의 전환 또는 다수의 전환과 프로필을 용융 보통 또는 높음을 선택합니다. </li><li> 선택 계산그룹화 섹션에서 그룹. </li><li> 파일 메뉴로 이동하고 결과를 저장하려면 저장을 클릭합니다. </li></ol></li></ol></li></ol>

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저자가 공개하는 게 없다.

르마와 함께 PCR은 제브라 피쉬의 유전자형을위한 강력한 기술입니다. 이 방법의 장점은 그것의 속도, 신뢰성, 및 심지어 점 돌연변이를 검출 감도이다. 전체 프로토콜은, 지느러미 클립에 용융 곡선 분석에서, 한 개인에 의해 8 시간 이내에서 수행 할 수 있습니다. 또한, 기술은 높은 처리량 스크리닝 의무이다 티듐 브로마이드의 사용을 필요로하지 않으며, 오염 문제를 최소화 할 수있는 모든 PCR ?...

제브라 피쉬 (다니오 rerio)는 널리 개발 및 질병 모델링 연구에 사용되는 척추 동물 모델 시스템입니다. 최근 다수의 형질 전환 및 돌연변이 기술은 제브라 피쉬 위해 개발되었습니다. 일반적 Tol2 트랜스포존 시스템 (1)에 기초하여 피드 형질 전환 기술은, 다 DNA 단편 조립체이 향상된 복제 옵션과 결합되었다. 아연 핑거 뉴 클레아 제 (ZFNs) 및 전사 활성제와 같은 이펙터 클레아 제 (TALENs)는 제브라 피쉬 3,4 모두 체세포와 생식 세포의 유전자 좌를 대상으로 사용되어왔다. 이 기술은 효율적으로 고주파 돌연변이 생성 및 세균 선 전송 3,4로, 유전자 변형 동물을 생성 할 수 있습니다. 이러한 발전에도 불구하고, 제브라 피쉬의 전통 유전자형 기술은 돌연변이 유발 및 형질 전환 도구의 모든 기능을 제한합니다. PCR은 때때로 restrictio 결합, 전기 영동 한 다음N 효소 소화가 널리 유전체 변형을 검출하기 위해 사용되지만, 시간 소모 및 작은 삽입 또는 삭제를 식별에 덜 민감하다. 의 TaqMan 프로브 분석은 높은 초기 비용을 가지고주의 최적화를 필요로합니다. PCR 제품의 시퀀싱은 며칠이 걸릴 대규모 검열을위한 실용적되지 않습니다 수 있습니다. 제한 단편 길이 다형성 (RFLP) 분석에만 제한 효소 인식 부위의 제한된 범위에 영향을주지의 SNP를 구별 할 수있다. 고해상도 녹는 분석 (르마), 폐 튜브 후 PCR 분석 방법은, 빠른 민감한, 저렴하고, 다수의 샘플을 심사하는 의무가 최근에 개발 된 방법이다. 르마는 SNP를, 돌연변이, 및 5-7 전이 유전자를 검출하는데 사용될 수있다. 르마는 이중 가닥의 DNA의 열 변성을 기준으로, 각각의 PCR 증폭 물이 특징 독특한 해리 (용융) 5을 가지고 있습니다. 샘플로 인해 T를 구별 할 수 있습니다O 서로 다른 염기 조성, GC 함량, 또는 길이는 일반적으로 형광 염료와 함께 만 이중 가닥 DNA (8)를 결합하는. 따라서, 르마는 다른 용융 곡선의 특성에 따라 서로 다른 유전자형을 구별 할 수 있습니다. 르마 저가의 시약을 사용하여 단일 단계 후 PCR 과정이므로, 높은 처리 전략을 위해 사용될 수있다. 르마 그래서 분석 다음 PCR 증폭 산물이 다른 애플리케이션에 사용될 수 있고, 비파괴이다. 르마 세포주, 마우스 및 인간 9-11 포함하여 많은 유기체 및 시스템에 적용되어왔다. 그것의 사용은 최근 징크 핑거 클레아 (ZFNs) 및 TALENs 6,12,13 의해 유도 돌연변이를 검출하기 위해 제브라 피쉬 설명되었다. 이 논문에서, 우리는 (그림 1) 배아와 성인 제브라 피쉬의 PCR 기반 르마을 수행하는 방법에 대해 설명합니다. 이 프로토콜은시를 포함하여, SNP를, 도입 유전자, 돌연변이를 검출하기에 적합하다ngle 기본 쌍 변경, 삽입, 또는 삭제.

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rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

제브라 피쉬는, 개발을 공부하는 질병을 모델링하고, 약물 검사를 수행하기위한 강력한 척추 동물 모델 시스템입니다. 최근 유전 도구의 다양한 돌연변이를 유도하고 유전자 변형 라인을 생성하기위한 여러 전략을 포함하여, 도입되었습니다. 그러나, 대규모 스크리닝은 시간 소모적이고 노동 집약적이다 전통적인 유전자형 방법에 의해 제한된다. 여기에서 우리는 고해상도 융점 분석 (르마)와 결합 PCR 기준 지브라 피쉬 유전자형을 분석하는 기술을 설명한다. 이 방법은 오염 유물의 낮은 위험과 함께, 빠른 민감하고 저렴합니다. 르마와 PCR에 의한 유전자형 높은 처리량 검사 프로토콜에 포함, 배아 또는 성인 물고기에 사용할 수 있습니다.

프로토콜 (작업의 흐름도는도 1에 도시 됨) 며칠 동안 하루 동안 수행 또는 단계로 분리 될 수있다. DNA 추출은 PCR 증폭 산물의 용해 및 분석을 따른다. 앰플 리콘의 용융의 온도는 크기와 GC-내용에 따라 다르지만 일반적으로 시작하고 50 ˚ C ~ 95 ˚ C의 최종 온도 (그림 2A와 2B)에 적합합니다. 용융물이 수행되면, 형광 용융 곡선의 분석은 일반적으로 사용 전후의 용융 영역을 ...

Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

고해상도 용융 분석 (르마)와 함께 PCR은 제브라 피쉬의 유전자형에 신속하고 효율적인 방법으로 보여줍니다.

고해상도 용융 분석과 PCR을 이용하여 신속하고 효율적인 Zebrafish의 유전자형

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

rapid-efficient-zebrafish-genotyping-using-pcr-with-high-resolution

Research

JoVE Journal

Biology

22.1K 조회수.  University of Utah School of Medicine.  고해상도 용융 분석 (르마)와 함께 PCR은 제브라 피쉬의 유전자형에 신속하고 효율적인 방법으로 보여줍니다. 

비디오: Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

시그마의 압축을 풉니다 - N - 앰프 조직 PCR 키트를 사용하여 마우스 조직의 신속한 Genotyping

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

연구

생물학

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Microscale 열 대류를 사용하여 신속한 PCR의 Thermocycling

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

에서 초기 단계의 배아의 효율적이고 신속한 분리<em&gt; 애기 장대</em&gt; 씨앗

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

높은 해상도 전체 마운트<em&gt; 제자리에서</em유전자 발현 및 기능을 연구하는 제브라 피쉬 배아에서&gt; 하이브리드

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

고해상도 이미징 및 개인 별이 미세 소관 역학의 분석 신진 효모에서

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...