저자는 Arabidopsis 식물의 유지 보수를 위해 양 제니퍼 M. Lommel와 타라 N. Rognan 감사합니다. 이 작품은 건강 보조금 1R15GM086846 - 01와 W. 샤오에 3R15GM086846 - 01S1의 세인트 루이스 대학과 국립 연구소에서 시작 기금에 의해 지원되었다.

I. 유전자 크로싱 1. 여성 부모 Emasculating DNA 시퀀스 다형성, 두 개의 다른 ecotypes 예 : 콜럼비아 - 0 (콜 - 0) 및 Ler가, 여성과 남성의 부모로 선택됩니다를 사용하여 모자 아버지 대립 유전자를 식별하기 위해. 식물 젊고 건강해야합니다. 하나는 바이저, 또는 육안을 확대, 해부 현미경을 사용하여 여성의 부모를 거세된 수 있습니다. 무대 - 12 꽃을 찾아 (스미스 외., 1990)와 가위와 작은 꽃자루의 기반을 클리핑하여 꽃이나 siliques 위와 아래를 제거합니다. 포셉에있는 꽃가루 곡물을 제거하고뿐만 아니라 꽃가루를 죽일 것입니다 95 %의 에탄올의 비커로 부드럽게 팁의 기반을 찍기로 집게를 소독. 부드럽게 집게를 사용하여 꽃 봉오리를 분리 캐내려고하고, 부드럽게 암술은 베어하고 그대로두고, 4 sepals, 4 꽃잎, 6 stamens를 제거합니다. 이 과정에서 carpels에 손상을 피하기 위해보십시오. 2. 꽃가루 기증자를 따기와 수분을 수행 최고의 꽃가루의 기증자는 꽃가루가 많이 흘리기있는 암술 (스미스 외., 1990)에 90 ° 각도로 확장 꽃잎과 함께 단계 14에서 열린 꽃 anthesed 있습니다. 기지에서 꽃을 잡고 그냥 작은 꽃자루 위에, 이것은 꽃이 열려 확산하게됩니다. 꽃밥과 준비 암술의 오명을 먼지. 수분 후, 오명은 쉽게 현미경으로 관찰할 수있는 노란색 꽃가루와 함께 다룹니다. 3. 십자가를 라벨링 식물의 모든 emasculated pistils을 pollinating 후, 우리는 보석 태그와 여성과 남성의 부모 및 날짜 정보와 함께 십자가를 라벨. 공장 주변 토양의 지분을 장소 지분에 꽃이 핌의 줄기를 묶어하는 문자열을 사용하고 비닐 봉지로 pollinated pistils을 커버. II. Arabidopsis의 배젖 조직의 분리 1. 준비 자료 액체 질소 탱크, 액체 질소, 해부 현미경, 미세 팁 포셉 (더몬트 생물학 5 INOX FST, 스위스.) 현미경 유리 슬라이드의 두 쌍 (우리는 보통 배젖 및 배아 조직을 수확하기 전에 준비가 필요한 자료를 얻을 3 &quot;X 1&quot;X 1.0 mm), sorbitol 0.3 M의 산도 5.7 솔루션 5 MM MES. 2. 수집 siliques 7 - 또는 8 - 일 - - 수분 후 (DAP), 씨앗이 embryogenesis의 후반 단계 어뢰 중순에 수확하고 배젖 및 배아에 대한 해부를 준비하고 있습니다. emasculated 꽃이 너무 어린 경우에는 때로는 90-10 DAP이 걸릴 수 있습니다. 3. 분리 배젖 및 배아 Arabidopsis 씨앗은 작은 있기 때문에, 우리는 배젖 및 배아를 분리 해부 현미경을 사용합니다. 현미경 8 DAP의 silique 넣고, silique 작은 꽃자루를 잡아 포셉 한 켤레를 사용하여 두 carpels 퓨즈 여백에 열려 silique을 슬라이드 포셉 또 한 쌍의의 팁을 사용합니다. sorbitol 0.3 M 5 MM MES의 산도 5.7 솔루션에 한 종자를 데리러 포셉 한 쌍의를 사용하여, 배아를 슬라이드 micropyle 끝에 작은 상처를 씨앗 코트에서 배젖 및 별도의 배젖를 밀어 포경 끝을 스퀴즈 (기노 시타 외., 2004). 액화 질소에 별도의 microtubes로 배아와 배젖 놔. 우리가 10-15 siliques에서 배아 또는 배젖을 축적 때까지 이것을 계속하고 -80 ° C 냉동고에 튜브를 저장합니다. 어떤 경우에는, 배젖은 씨앗 코트, 즉 분리하실 필요가 없습니다, 하나는 유전자 각인 분석 배젖 및 종자 코트 혼합물에서 총 RNA를 분리하실 수 있습니다. III. Bisulfite 배열 1. 필요한 시약 , : 게놈 DNA, 제한 효소, 3 M NaOH (갓 만든), 6.42 M의 요소 / 4 M 나트륨 bisulfite를 (190 2 M 나트륨 metabisulfite, 시그마 - 알드리치, S9000, Na2S2O5, 분자량) 준비 Cetyltrimethyl 암모늄 브로마이드 (CTAB) 10 MM 히드로퀴논, DNA 정제 키트 (Promega 마법사 DNA 청소 시스템, 고양이. # A7280), TE 버퍼, 6.3 M NaOH (갓 만든), 10 M NH 4 OAc, 20 μg / μL tRNA 및 100 % 에탄올 . 2. bisulfite 치료 프로토콜의 세부 사항 <ol><li> 게놈 배젖 또는 CTAB 절차를 사용하여 배아 DNA (로저스 Bendich, 1988)을 분리. </li><li> 다이제스트 100 NG - 20 μL 100 분석하는 영역 이외의자를 제한 효소로 μL 총 볼륨의 게놈 DNA 2 μg. MEA의 발기인을 위해, 우리는 XhoI, NdeI 및 PstI이나 HindIII를 사용합니다. </li><li> 5 분 동안 DNA를 끓는하여 제한 효소를 변성 후 얼음 끄다. </li><li> 15 분 37 3 M NaOH와 부화의 9분의 1 볼륨 (20 μL 소화 DNA를 2.2 μL) ° C를 추가합니다. </li><li> 250 μL PCR 튜브에 대한 해결책을 전송합니다. </li><li> 멸균 증류수 10 ML에 요소의 7.5 g을 디졸브, 천천히 1~2시간 이상의 나트륨 metabisulfite의 7.6 g을 추가하고 가열은 일반적으로 해소하는 데 도움이, 갓 만들어진 10 M NaOH와 5 산도를 조정, 마지막에 살균 증류수를 추가 20 ML에 볼륨. 이것은 6.24 M의 요소 / 4 M 나트륨 bisulfite 솔루션입니다. </li><li> 각각 6.24 M의 요소 / 5.36 M의 최종 농도 4 M 나트륨 bisulfite 솔루션과 3.44 M을 (폴린 외., 1998) 추가합니다. 예를 들어, 6.42 M의 요소 / 변성 게놈의 DNA (샤오 외., 2003)의 위 22.2 μL 4 M 나트륨 bisulfite 솔루션의 208 μL를 추가합니다. </li><li> 0.5 MM (20 μL 소화 12 μL)의 최종 농도에서 DNA 10 MM의 히드로퀴논를 추가합니다. </li><li> . 55 ° 15 분 95 ° C 30 초 동안 C 30 사이클 : PCR 기계에서 bisulfite 치료를 실시 </li><li> Promega에서 마법사 DNA 청소 시스템을 사용하여 bisulfite 치료 DNA를 탈염하다와 (야콥센 외., 2000) 프로토콜을 따르십시오. </li><li> 탈염 후 열을에서 발견한 TE의 정확한 부피를 측정하고 37 0.3 M. 부화의 최종 농도 6.3 M NaOH를 추가 ° C를 15 분. </li><li> 최종 3 M의 농도 20 μg / μL tRNA 2 μL, 100 %의 에탄올 3 권 ~ 10 M NH 4 OAc (산도 7.0) 추가하고 다음 섞는다. 14,000 rpm으로 15 분 동안 원심 분리기. </li><li> , 70 % 에탄올로 한 번 펠렛을 씻어 짧은 원심 분리기를하고, 여분의 에탄올을 제거하십시오. </li><li> 25 5-10분 및 resuspend위한 speedvac에 건조 펠렛 - DNA의 양을 시작에 따라 100 μL TE 버퍼. 나트륨 bisulfite - 대우 DNA 지금은 PCR 분석을위한 준비가되었습니다. </li></ol> 3. PCR 증폭 <ol><li> unmethylated cytosines가 유라실로 변환되므로, 이것은 템플릿으로 bisulfite - 대우 DNA를 사용하여 큰 조각을 증폭하기가 어렵습니다. 따라서, 우리는 일반적으로 더 이상 500 BP보다 제품을 증폭하기 위해 primers를 디자인하지 않습니다. 순서 4 - KB MEA의 발기인을 위해, 우리는 primers 많은 세트를 설계하고 14 중복 조각이 전체 지역을 커버하기 위해 증폭 (샤오 외., 2003). </li><li> 순서 가기 - 스트랜드는 앞으로 입문서, I)를 설계 primers에 추가 긴 세포핵이없는 높은 어닐링 온도를 위해서는 G (구아닌)이 풍부한 지역을 선택, II) 변경 C (사이 토신) Y을 ( CG와 CNG 컨텍스트에서 pyrimidine) 및 T (thymine)에 남아있는 C를 변경합니다. 역방향 프라이머, I)을 설계에 C - 풍부한 지역을 선택, II) CG와 CNG 컨텍스트에서 R (purine)로 G를 변경하고 A (아데닌)에 남아있는 G를 변경합니다. </li><li> CG와 CNG 컨텍스트에서 R로 G를 변경하고 선택 역방향 프라이머, I)를 설계 A.에 남아있는 G를 변경 II); 순서 하단 스트랜드는 앞으로 입문서, I)를 설계에 C - 풍부한 지역을 선택 G - 풍부한 지역, II)는 CG와 CNG 컨텍스트에서 Y (pyrimidine)로 C를 변경하고 T.에 남아있는 C를 변경 </li><li> 우리는 일반적으로 각각의 PCR 증폭 (샤오 외., 2003)에 대한 템플릿으로 나트륨 bisulfite - 취급 DNA 1-2 μL를 사용합니다. PCR 제품은 다음 젤 삽입으로 정화하고 토포 TA 클로닝 벡터 pCR2.1 (Invitrogen)에 복제하는 조각의 정확한 크기를 확인하기 위해 젤 전기 영동으로 분석해야합니다. 하나의 식민지가 데리러와 교양되며 플라스미드 DNA가 추출 및 시퀀싱을위한 보냈습니다. </li></ol> 4. 서열 분석 bisulfite 배열의 원칙은 unmethylated 사이 토신은 PCR 제품의 thymine으로 증폭됩니다 pH5.0에서 나트륨 bisulfite의 높은 농도에 의해 가수 분해 deamination에 의한 유라실로 변환됩니다이며 5 메틸 사이 토신은 나트륨 bisulfite에 의해 수정되지 않습니다 반면 과 PCR 증폭 (.; Frommer 외, 1992. 클라크 외, 1994) 이후 사이 토신으로 남아 있습니다. 시퀀스 결과를 얻기 후에, 우리는 PCR의 증폭에 사용되는 스트랜드 특정 템플릿과 그것을 비교하십시오. 템플릿의 사이 토신 잔여물이 시퀀싱 결과 thymine으로 글을 읽는다면, 그것은 사이 토신이 methylated되지 않았음을 나타냅니다. 템플릿의 사이 토신 잔여물이 순서에있는 사이 토신 남아있을 경우, 그것은 사이 토신이 methylated는 것을 의미합니다. IV. 대표 결과 <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="/ 그림 1" /> 그림 1.

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관심 없음 충돌 선언하지 않습니다.

그것은 배젖 및 종자 코트에서 배아를 분리하는 것이 비교적 쉽기는하지만 embryogenesis의 초기 또는 중간 단계에서 어뢰 특히 씨앗을 위해, 종자 코트에서 배젖를 구분 지루합니다. 종자 코팅은 일부 유전자에 대한 조직의 매우 작은 금액을 기여 때문에, 예를 들어, MEA와 FWA, 우리는 종자 코트에서 배젖를 구분하지 않아도됩니다. 우리가 배젖 및 종자 코트 조직의 혼합물에서 RNAS을 분리할 수?...

용품 <ul><li> 해부 현미경 </li><li> 가위 </li><li> 파인 팁 집게 </li><li> 쥬얼리 태그 </li><li> 플랜트 스테이크스 </li><li> 문자열이나 트위스트 - 넥타이 </li><li> 4 &quot;X 2&quot;X 8 &quot;폴리에틸렌 가방 </li><li> 3 &quot;X 1&quot;X 1.0 mm의 현미경 슬라이드 </li><li> 액체 질소 </li><li> 액체 질소 용기 </li><li> 열 차단 </li><li> PCR 튜브 </li><li> Thermocycler </li><li> Microcentrifuge 튜브 </li><li> Microcentrifuge </li><li> 겔 전기 영동 시설 </li><li> Arabidopsis thaliana 콜럼비아 - 0 식물 </li><li> Arabidopsis thaliana 랜즈 erecta 식물 </li></ul> 시약 <ul><li> 70 % 에탄올 </li><li> 95 % 에탄올 </li><li> 100 % 에탄올 </li><li> Sorbitol 0.3 M 5 MM MES - 산도 5.7 </li><li> Cetyltrimethyl 질산 브로마이드 (CTAB) </li><li> 100 % 에탄올 </li><li> Cholorform </li><li> 제한 효소 </li><li> 3 M NaOH </li><li> 6.3 M NaOH </li><li> 6.24 M 요소 / 4 M 나트륨 Bisulfite </li><li> 멸균 증류수 H 2 O </li><li> 10 MM의 히드로퀴논 </li><li> 마법사 DNA 청소 시스템 (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 μg / μL tRNA </li><li> TE 버퍼 </li><li> 토포 TA 복제 키트 (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana은 epigenetic 메커니즘을 연구를위한 훌륭한 모델 생물이다. 그 이유 중 하나는 DNA의 methyltransferases의 손실 함수 NULL의 돌연변이가 따라서 게놈에있는 DNA의 methylation의 손실이 성장과 발전에 미치는 영향 연구하는 시스템을 제공하고, 실용적입니다. 각인은 모자 아버지의 대립 유전자의 미분 표현을 나타냅니다과 포유 동물과 식물 모두에서 복제 개발에 중요한 역할을합니다. DNA의 methylation은 각인 유전자의 모자 또는 아버지 대립 유전자가 표현하거나 침묵 여부를 결정하는 것이 중요합니다. 꽃 식물에서, 복제에서 더블 수정 이벤트가있다 : 한 정자 세포는 배젖을 일으키다 수있는 중앙 세포와 배아 그리고 두 번째 정자 퓨즈를 형성하기 위해 난자를 fertilizes. 배젖은 각인이 공장에서 발생하는 조직입니다. 메데아, SET 도메인 Polycomb 그룹 유전자 및 FWA, 꽃을 조절 전사 인자는 배젖 및 표현에 인쇄되는 표시된 처음 두 유전자 아르의 DNA의 methylation과 demethylation에 의해 제어됩니다 식물. 유전자와 배젖의 methylation 패턴의 각인 상태를 결정하기 위해, 먼저 배젖를 분리 수 있어야합니다. 씨앗 Arabidopsis에서 작은 있기 때문에, 그것은 Arabidopsis 배젖를 분리하고 methylation을 확인하기 위해 도전 남아 있습니다. 이 비디오 프로토콜에서는, 우리는 씨앗에서 배젖 조직을 분리, 유전자 십자가를 실시하고, bisulfite의 시퀀싱에 의해 methylation 상태를 확인하는 방법을보고합니다.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

각인은 식물과 동물의 복제에 현상입니다. DNA의 methylation은 각인의 메커니즘에서 중요한 역할을합니다. 배젖를 분리 및 각인 유전자의 methylation 상태를 결정<em&gt; Arabidopsis</em&gt;는 어려울 수 있습니다. 이 프로토콜에서는, 우리는 배젖를 분리하고 bisulfite의 시퀀싱에 의해 methylation을 결정하는 방법에 대해 설명합니다.

에 각인 유전자의 DNA Methylation의 결정 Arabidopsis 배젖

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K 조회수.  Saint Louis University. 각인은 식물과 동물의 복제에 현상입니다. DNA의 methylation은 각인의 메커니즘에서 중요한 역할을합니다. 배젖를 분리 및 각인 유전자의 methylation 상태를 결정 Arabidopsis는 어려울 수 있습니다. 이 프로토콜에서는, 우리는 배젖를 분리하고 bisulfite의 시퀀싱에 의해 methylation을 결정하는 방법에 대해 설명합니다.

비디오: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

의 DNA Methylation : Bisulphite의 변경 및 분석

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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생물학

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

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

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

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

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

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

유전자 탐구하는 종피 침구 분석<em&gt; 체외</em&gt; 어떻게 배젖 컨트롤 종자 발아에<em&gt; 애기 장대</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

체외 메틸화 분석에서 단백질 아르기닌 메틸화를 공부합니다

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

자료 쌍 해상도에서 DNA 메틸화의 평가에 대한 표현 바이 설 파이트 시퀀싱을 감소 향상

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

유전자 특이 발현과 DNA 메틸화 변화와 성상 세포의 전사 활동의 상관 관계<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA 메틸화 분석 차세대 시퀀싱 대상

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...