도움이 의견을 거스 - 절대 안전한 프로토콜 Paja Sijacic과 코트니 Hollender를 공유하기위한 거스와 그림 1, Detlef 위겔의 사진을 : 우리는 pTSO2 구축을위한 Chunxin 왕 감사합니다. Z. L S 실험실에서 연구는 미국 국립 과학 재단 (IOB0616096 및 MCB0744752)에 의해 지원됩니다. ZL는 부분적으로 메릴랜드 농업 실험 역 대학에 의해 지원됩니다.

I. 꽃 - 물놀이 Arabidopsis thaliana의 변화 <ol><li> 이전 변환에 약 1 개월, 냄비에 약 10-15 식물 4-8 냄비 (5 인치 사각형 냄비)에 Arabidopsis 씨를 뿌리다. 정상 다산과 식물, 플라스미드 구조마다 식물의 4 냄비는 충분합니다. 하나는 감소 다산과 돌연변이 Arabidopsis를 변환하는 경우, 그에 따라 냄비의 수를 증가시킵니다. </li><li> 식물은 표준 약관 (22 16 시간 조명과 어두운 8 시간 ° C)에서 재배하고 있습니다. 식물은 짧은 하루 더 낮은 온도 하에서 재배하는 경우, 그들은 변화를 위해 준비하는 꽃 이상으로 오래 걸립니다. 좋은 식물 관리는 성공적인 변화를위한 필수적인 건강한 식물을 보장합니다. </li><li> 식물은 나올와 꽃으로 시작하는 경우. 그들은 지금 변화를위한 준비가되어 있습니다. 변환 효율을 향상시키기 위해, 3-4일 변환하기 전에, 곧 그들은 더 많은 보조 촬영 형성을 장려하기 위해 나올 필요로 메인 꽃이 핌 골인을 잘라. 식물 변환 전날 물. 이것은 토양이 꽃의 수영하는 동안 너무 많이 침투 미디어를 흡수하지 않을 보장합니다. </li><li> Agrobacterium tumefaciens 변형 GV3101은 pTSO2를 들고와 (거스, 50 μg / ML Kanamycin : pTSO2의 경우) : 이틀 전에 변환하기 위해, 적절한 항생제를 포함한 40ml LB의 예방 : 거스는 건설. pTSO2 : : 거스는 Kanamycin 3 저항을 수여 NPTII 유전자를 가지고 pBIN20 벡터에 복제됩니다. 28-30 ° C.에 떨고 배양기에서 하룻밤 성장 </li><li> 그 다음날, 50 μg / ML Kanamycin이 포함된 LB의 400ml에 밤새 문화의 전송 8ml. 28-30 ° C.에 떨고 배양기에서 하룻밤 성장 </li><li> 변화의 날, Agrobacterium 문화의 OD 600 약 0.8 (0.5 to1 사이 OD 600 허용된다)에 도달하면, 5000rpm 8 분에서 Agrobacterium 야간 문화를 스핀 다운. </li><li> 침투 미디어 1 L에있는 Agrobacterium 펠렛을 (리터) (10mM MgCl 2, 5 %의 자당, 0.44mM 6 benzyladenine (BA), 0.3 % silwet L - 77, 1X Gamborg의 비타민 솔루션과 autoclaved 물). Resuspend 매체 autoclaved해야하지만 신선한 만들 필요하지 않습니다. 접시 (예 : 8로 &quot;x15&quot;X2 &quot;Pyrex 유리 접시)에 1 L Agrobacterium 함유 침투 매체를 붓고. </li><li> 냄비 반전 (식물에서 떨어지지 않을 것이란)와 침투 솔루션에 식물의 모든 공중 부분을 찍어, 한 번에 한 오분 냄비 기다. 공장의 모든 항공 부분이 침투 매체에 열중하는 동안, 토양은 토양에 곰팡이 성장의 기회를 줄이기 위해 침투 미디어 중 하나를 흠뻑 젖어셔서 마십시오. </li><li> 마른 후, 트레이에 종이 타월의 여러 레이어에서 자신의 측면에서 모든 담근 판돈을 놓으십시오. 종이 타올은 침투 미디어의 접근 금액을 만끽해보세요. 높은 습도를 보장하기 위해 플라스틱 포장으로 트레이를 다룹니다. 식물 성장 챔버에 트레이를 놓습니다. </li><li> 다음날, 똑바로 플라스틱 랩, 종이​​ 수건, 장소의 냄비를 제거합니다. 4-5 더 많은 일 동안이 식물을 물을 마십시오. 이후, 그들은 씨앗을 설정할 때까지 정상적으로 식물을 유지하고 대량의 종자를 수집합니다. </li><li> 4g의 멋진 한천, 압력솥, 그리고 추가 1M NaOH와 5.8-500 ML의 물, 산도에 용해, MS 매체가 50 μg / ML Kanamycin (중량 2.2g Murashige가 포함된 비타민없이 기초 소금을 Skoog와 함께 접시 (150x15mm 페트리 접시)을 부어 ) 50 μg / ML 최종 농도 Kanamycin를 추가합니다. 번호판이 필요 ° C까지 4 보관됩니다. </li><li> 항생제 - 내성 유전자 변형 식물을 선택하기 위해, 하나는 최소한 20,000 씨앗을 화면으로 필요합니다. 0.1 그램은 약 5,000 씨앗입니다. 무게와 씨앗의 양을 예상됩니다. </li><li> 30 초 동안 70 %의 에탄올과 15 ML 팰컨 튜브를 세척하여 씨를 소독. 한번 무균 물로 씨앗을 씻으십시오. 가게에서 구입 5 % 표백제 (Clorox)의 1시 10분 희석이 들어있는 용액에 30 초 동안 씨앗을 만끽해보세요. 멸균 물 3-6 시간을 씨를 린스. 무균 후드, 각 MS (Kanamycin) 플레이트에 피펫 5000에 대한 씨앗은 오염을 피하기 위해 Micropore 3M ​​테이프로 번호판을 봉인, 접시에 균일하게 씨앗을 확산. </li><li> 4 번호판을 품어 ° C 3-4 일 동안 어둠 속에서, 다음 챔버 (같은 식물 성장 챔버 수)에 접시를 전송합니다. 약 14 일 후, 유전자 변형 모종 녹색 유지하지만 이외의 유전자 변형 것들은 창백 돌려 죽어 가고 있습니다. 둔화가 매체에서 뿌리를 철수하고 토양에서 그들을 배치하여 토양에 유전자 변형 묘목을 전송합니다. </li></ol> II. pTSO2을 검토 : : 거스 표현 패턴 <ol><li> 수확 pTSO2 : 거스 형질 전환 모종 및 유리 섬광 약병이나 microfuge 튜브의 감기 90 % 아세톤에 배치그것은 얼음에 보관됩니다. 한 샘플의 큰 숫자를 분석하는 경우 폴리스티렌 microtiter 플레이트 (안 폴리 프로필렌)도 사용할 수 있습니다. </li><li> 모든 샘플이 채취 후 20 분 상온에서 튜브를 놓으십시오. 이 시간 동안 X - Gluc (0.2 % 트리톤 X - 100, 50mM NaHPO 4 버퍼 pH7.2, 2mM 칼륨 Ferrocyanide, 2mM의 칼륨 Ferricyanide)와 얼음에 장소없이 신선한 얼룩 버퍼를합니다. </li><li> 샘플에서 아세톤을 제거하고 얼룩 버퍼를 추가합니다. </li><li> X - Gluc 얼룩 솔루션 (0.2 % 트리톤 X - 100, 50mM NaHPO 4 버퍼 pH7.2, 2mM의 칼륨 Ferrocyanide, 2mM의 칼륨 Ferricyanide, 2mM X - gluc)를 구성합니다. 샘플에서 얼룩 버퍼를 제거하고 샘플을 X - Gluc을 포함하고있는 얼룩 버퍼를 추가합니다. </li><li> 15-20분위한 진공 아래 샘플을 침투. 천천히 진공을 해제하고 모든 샘플이 얼룩 솔루션의 표면 아래 싱크되었는지 확인합니다. 필요한 경우 진공이 릴리스되면 모든 샘플 싱크까지 침투를 반복합니다. </li><li> 37 품어 샘플 ° C X - Gluc와 얼룩 버퍼에 하룻밤. </li><li> 인큐베이터에서 샘플을 제거하고 얼룩 버퍼를 제거합니다. 삼십분마다 변경 상온에서 (20 %, 35 % 및 50 % 에탄올) 연속 에탄올 시리즈의 샘플을 씻으십시오. </li><li> 실온에서 30 분 FAA 정착액 (50 % 에탄올, 5 % 포름 알데히드 10 % 초산, 나머지 물)에 조직을 수정. FAA를 제거하고 70 % 에탄올을 추가합니다. 이 시점에서 조직은 시각 수 있으며 디지털 카메라가 장착된 해부 현미경 사진. TSO2가 (그림 1) 표현이 어디 진한 파란색 색상을 나타냅니다. 또한, 조직은 ° C 나중에 볼 수 있도록 4 저장할 수 있습니다. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="그림 1" /> 그림 1 pTSO2 :. : 유전자 변형 모종의 거스 표현 (A), 측면 뿌리 (B), 그리고 루트 팁 (C). TSO2 발기인은 젊은 분열 조직에서 매우 활성화되어 있습니다. (C)와 같이 산발적 표현식 패턴은 특정 세포주기의 단계에있는 유전자 표현의 전형이다. </li></ol> 대표 결과 제대로하면, 변환 효율은 약 0.1-0.2 %되어야합니다. 다른 단어에서, 하나는 모든 5,000 종자를 선별하여 50-10 형질 모종을해야합니다. 평균 약 100 유전자 변형 라인은 야생 형 식물의 4 냄비를 변환하여 얻을 수 있습니다. 에 대한 pTSO2를 포함하는 유전자 변형 모종 : 거스 구축 3, 거스 활동을 반영 진한 파란색 색상 어린 나뭇잎, 쏴, 아펙스, 루트 팁, 그리고 측면 루트 primordia (그림 1)을 포함하여 적극적으로 나누어 세포에서 발견된다. 비 균일한 산발적 패턴은 세포주기 단계 - 구체적인 표현의 특징입니다.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

변환 효율은 아래 설명하는 여러 가지 요인에 의해 결정됩니다 : <ol><li> 식물의 건강과 자신의 나이가 기본 중요합니다. 다수의 미숙 꽃 봉오리를 생산 약 한 달쯤 식물 이상적입니다. 변환하기 전에 보조 촬영 형성 3-4일 권장하는 기본 골인을 트리밍하는 것은 변환 효율을 증가합니다. 이전 식물 transformants을 일으키다하지만 낮은 비율로합니다. </li><li> 식물의 불임은 중요하다. 하나는 감소 다...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>6-benzyladenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>852430</td>
<td>&nbsp;</td>
<tr>
<td>Silwet-77</td>
<td>&nbsp;</td>
<td>Crompton Corp.</td>
<td>&nbsp;</td>
<td>Toxic, wear gloves</td>
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<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
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<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
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<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
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floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

유기체로 외국 유전자를 도입하는 능력은 현대 생물학과 생명 공학에 대한 기초입니다. 모델 꽃 공장에서<em&gt; Arabidopsis thaliana</em&gt; 꽃 - 딥 변환 방법<sup&gt; 1-2</sup&gt; 때문에 간단, 효율성 및 저렴한 비용의 이전의 모든 방법을 대체하고 있습니다. 젊은 꽃의 특히, 골인<em&gt; Arabidopsis</em&gt; 식물의 솔루션에 담근 아르<em&gt; Agrobacterium tumefaciens</em&gt; 구체적인 플라스미드 구조를 들고. 마른 후, 식물이 정상적인 성장과 항복 씨앗, 외국 유전자로 변환하고 항생제를 포함하는 매체에 대해 선택할 수있는 작은 비율로 반환됩니다. 크게 촉진이 꽃 - 물놀이 방법<em&gt; Arabidopsis</em&gt; 연구와 식물 유전자 기능에 대한 우리의 이해에 크게 공헌했습니다. 본 연구에서는, 우리는 기자의 유전자를 변형하는 꽃 - 수영 방법을 사용<em&gt; β - glucuronidase (거스)</em&gt;의 통제하에<em&gt; TSO2</em&gt; 발기인.<em&gt; TSO2</em&gt; Ribonucleotide 환원 효소 (RNR) 작은 subunit에 대한 코딩<sup&gt; 3</sup&gt; 세포주기의 S - 단계에서 dNDP 생합성에 필수적인 세포주기 규제 유전자입니다. 심사<em&gt; 거스</em유전자 변형&gt;의 표현<em&gt; Arabidopsis</em&gt; 모종가를 보여줍니다<em&gt; TSO2</em&gt;를 적극적으로 조직을 나누어 표현입니다. 보고된 실험 방법 및 자료를 쉽게 고등학교 및 대학 수준의 연구뿐만 아니라 교육뿐만 아니라 적용할 수 있습니다.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

이 문서의 꽃 - 수영 방법을 보여줍니다<em&gt; Agrobacterium tumefaciens</em의&gt; - 중재 변환<em&gt; Arabidopsis thaliana</em&gt;. 세포주기 조절 발기인 - 기자를 소개함으로써,<em&gt; pTSO2 : β - glucuronidase (거스)</em&gt;로<em&gt; Arabidopsis</em&gt; 우리는 하나가 유전자 변형 모종의 거스 기자 표현식을 감지하는 방법을 보여줍니다.

의 꽃 - 물놀이 변환 Arabidopsis thaliana 검사 pTSO2 : β - glucuronidase 리포터 유전자 발현

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

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Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

36.0K 조회수.  University of Maryland College Park. 이 문서의 꽃 - 수영 방법을 보여줍니다 Agrobacterium tumefaciens - 중재 변환 Arabidopsis thaliana. 세포주기 조절 발기인 - 기자를 소개함으로써, pTSO2 : β - glucuronidase (거스)로 Arabidopsis 우리는 하나가 유전자 변형 모종의 거스 기자 표현식을 감지하는 방법을 보여줍니다.

비디오: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

의 조직 화학 염색<em&gt; 애기 장대</em&gt; 차 전지 벽 요소

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

아마의 꽃 - 딥 변환 (<em&gt; LINUM usitatissimum</em&gt;) 높은 변환 속도와 형질 전환 자손을 생성합니다

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

컨포칼 이미징을 통한 ERL1 동역학의 시공간 조사

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

저자 스포트라이트: 기공 계통 세포에서 막 이동 이벤트를 연구하기 위한 이미지 기반 방법  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

기공 계통 세포에서 막 트래피킹 사건을 연구하기 위한 이미지 기반 방법

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...