우리는 배아 분리에 대한 자신의 조언을 탈 Nawy 마틴 바이어에게 감사의 말씀을 전합니다. MTR, VG, UG와 CB는 배아 분리 장비를 고안했다. MTR, VG와 CB는 배아 분리 프로토콜을 개발했다. MTR, VG와 CB는 프로토콜을 확립 배아를 격리하고, 생성 된 배아의 cDNA, VG는 PCR, MTR GUS 염색, JJ 물고기 실험을 수행 하였다. MTR, VG, CG 및 UG는 원고를 썼다. 이 작품은 취리히 대학교, 로슈 연구 재단 (MTR까지)의 친목 및 스위스 국립 재단 (UG와 CB까지)에서 교부금에 의해 투자되었다.

절차는 그림 1의 순서도에 요약되어 있습니다. microcapillaries과 악기 설정은 그림 2와 그림 3에 표시되고, 배아 분리의 일반적인 단계는 그림 4에 표시됩니다. 1. 재료 및 버퍼 준비 유리 microcapillaries 1.1 실리콘 코팅 <ol><li> Sigmacote (시그마)의 ~ 5 ml의 15 ML 팔콘 튜브에 microcapillaries를 놓고 여러 번 반전. </li><li> 용액을 제거, 60 ℃에서 알루미늄 호일에 모세 혈관을 포함 팔콘 튜브를 배치하고 3 시간을 위해 그들을 구울 상온에서 보관하십시오. </li></ol> 1.2 ~ 50-100 μm의 직경 microcapillary 정보를 얻을 <ol><li> 수동 분젠 버너를 통해 또는 상용 풀러 (수직 필라멘트 끌어 당기는 사람 또는 micropipette의 풀러)를 사용하여 하나 1mm 직경의 유리 모세관을 당기십시오. </li><li> 다이아몬드를 사용팁 펜이나 원하는 구멍을 만들 뽑아 모세관의 끝 부분을 잘라 블레이드. 스테레오 현미경 제일 모양의 모세 혈관을 선택합니다. 오프닝은 50 ~ 100 μm의 (그림 2)이어야한다. </li></ol> 1.3 슬라이드 준비 <ol><li> 5 분 Sigmacote (~ 1 ML / 슬라이드) 모든 우물을 커버하여 깨끗한 슬라이드를 Siliconize, 제거합니다. 알루미늄 호일 빵 3 시간. 상온에서 보관하십시오. </li><li> 10 % SDS, nuclease가없는 물에 2 × 2 분 (DEPC-DDH 2 O를 압력 가마로 소독), 70 % 에탄올에서 2 분, 100 % 에탄올에 2 분에서 10 분 동안 다 잘 유리 현미경 슬라이드를 씻어 공기 건조. 모든 단계는 멸균 코 플린 단지에서 수행됩니다. 슬라이드는 각각의 사용과 철저한 청소를 제공하는 여러 번 다시 사용할 수 있습니다. </li><li> 직전 배아 분리 확산 ~ 10 0.5 μL MG / ML 소 혈청 알부민 (BSA) 각 잘 공기 건조의 전체 표면에 피펫. </li></ol> 1.4 현미경 및 모세관 설정 &lt;OL&gt; <li> 10X 및 20X 배율 목적으로 거꾸로 현미경을 사용합니다. 빛의 명암을 최적화 (배아는 매우 투명). </li><li> 현미경 옆에있는 모세관 유리를 보유하는 micromanipulator에 놓습니다. 모세관 유리 고무 튜브를 통해 microinjector (그림 3A) 또는 일반 P-200 피펫에 연결되어 있습니다 (그림 3B,이 설정에 대한 자세한 설명은 설명을 참조하십시오). </li><li> ~ 70 ° 각도에서 현미경 슬라이드 (그림 3) 위의 모세 혈관을 배치하고 시야의 구멍을 가지고 위치를 조정합니다. </li><li> 직전 배아 분리를 차지 해제 ~ ~ 10 ㎕의 BSA (1 MG / ML) 코트 모세관을합니다. </li></ol> 1.5 버퍼 표 1은 다운 스트림 응용 프로그램에 따라 분리 및 대상 버퍼를 보여줍니다. <ol><li> ~ 샘플 당 1 ML 절연 버퍼 갓 전에 사용하고 얼음에 보관 준비. </li><li> 얼음 (분자 응용 프로그램) 또는 습기 챔버 현미경 슬라이드 (세포 학적 응용 프로그램)에서 0.5 ML Eppendorf의 낮은 바인딩 튜브 대상 버퍼를 준비합니다. </li></ol> 2. 씨앗 해부 및 배아 추출 종자 개발 2.1 동기화 <ol><li class="jove_content"> 꽃 거세와 다른 꽃과 노출 암술의 접촉을 피하면서 그 성장 챔버 2 일 보관 후, 그들 (예 : 11) 수분. </li><li class="jove_content"> 취소 씨의 현미경 조사하여 성장 조건에 따라 개발 단계를 테스트합니다. 4 DAP는 구상 굴복 - 우리의 성장 조건에 씨앗 (DAP) 수분 후 2.5 일 수집 (21에서 16 시간 빛 ° C, 18, 어두운 8 시간 ° C 및 70 % 습도) 주로 2-4 배아와 씨앗은 3.5 수집 나왔고 배아. </li></ol> 2.2 씨의 해부 및 파열 <ol><li> 의 제거를포셉과 인슐린 바늘 입체 현미경 하에서 15 siliques (~ 2.5 DAP)에서 eeds. </li><li> 얼음에 배치 2 ㎖ 둥근 바닥 에펜 도르프 튜브에 20 ㎕의 절연 버퍼에 씨앗을 담가. </li><li> 조심스럽게 씨앗 추출물 흐린 때까지 배아를 풀어 플라스틱 유봉 (DEPC-DDH 2 O로 세척, 10 % SDS로 미리 청소하고 70 % 에탄올로 세척)에 씨앗을 분쇄. 적용 할 힘은 재판시 모든 사용자에 의해 결정되어야한다. </li><li> 유봉을 씻어 샘플을 희석 분리 버퍼 300 μL와 유봉을 씻어. </li><li> 스핀 다운 5 초 동안 5,000 XG에서 추출합니다. 부드럽게 위아래 2 ~ 3 번 pipetting하여 펠렛 추출물을 resuspend을. </li><li> 30 μm의 나일론 메쉬 (PartecCelltricks에서 예를 들어, 튜브 어댑터에 장착)와 추출물을 필터링합니다. 추가 200 μL 절연 버퍼 메쉬를 씻어. </li></ol> 3. 배아 분리 3.1 슬라이드 준비 <ol> &lt;리&gt; 장소 거꾸로 현미경의 무대에 깨끗하고, BSA 코팅 및 실리콘 처리 다 잘 슬라이드 위아래로 부드럽게 pipetting하여 필터링 씨 추출물을 resuspend을 1 또는 2 우물에 40-50 μL 씨 추출물 2 방울을 피펫 . 한 번에 1 ~ 2 방울을 선별하여 샘플의 증발을 방지합니다. </li><li> 이전으로 준비 다른 슬라이드의 잘에 신선한 격리 버퍼의 장소 50 μL (1 차 세척 드롭). 증발을 방지하기 위해 덮인, 습기 챔버에이 슬라이드를 유지합니다. </li></ol> 3.2 스크린, 청소, 수집 <ol><li> 10X 배율 목적으로 원하는 단계에서 배아 종자 추출물의 방울을 화면. 필요한 경우, 20 배 배율로 단계를 확인합니다. 태아는 일반적으로 슬라이드의 바닥에 가라. </li><li> 수동 텅스텐 바늘, 인슐린 바늘 또는 이와 유사한 장비와 배아 주위에 파편을 제거합니다. </li><li> micromanipulator에, T를 사용하여 배아 근처 모세관 유리를 이동가능한 적은 솔루션으로와 배아까지 아케. </li><li> 여러 개의 배아를 (예를 들어, 모두 한 방울의) 수집 및 1 차 세척 드롭 (분자 응용 프로그램) 또는 대상 버퍼 (세포 학적 응용 프로그램)에서 그들을 놓습니다. 각 수집 라운드 최소 볼륨 (모든 수집 라운드의 총 볼륨이 &lt;5 μL이어야한다)에서 수집해야 5-10 분, 배아 내에서 유지되어야한다. </li><li> 배아의 원하는 양 (중앙, 가능하면 기억을 용이하게하기 위해) 1 차 세척 드롭 수집 될 때까지 검사와 컬렉션을 반복합니다. </li><li> 세척 드롭 (파편이 이월 된 경우, 기억하기 전에 바늘을 제거)에서 한 번에 모든 배아를 채취. </li><li> 50 μL의 2 차 세척 드롭으로 배아를 놓습니다. 3.2.6 반복합니다. </li><li> 대상 버퍼에 배아를 놓습니다. 전송은 모세관 끝의 유일한 접촉, 그리고 침수를 포함해야합니다. </li><li> microcapil를 교체다음 샘플을 위해 라히. </li></ol>

<ol>
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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

우리는 효과적인 급속 배아 분리 프로토콜을 개발, 쉽게 다른 실험실로 전송 될 수 있습니다. 여기에 설명 된 장비는 거꾸로 현미경, 미세 조작기, 유리 microcapillaries, 수직 필라멘트 풀러와 microinjector (그림 3A)으로 구성되어 있습니다. 설치가 전사 체학 분석 17 한 동물 세포 분리에 대해 설명한 것과 비슷합니다. 우리는 또한 성공적으로 유리 microcapillaries?...

현화 식물의 배아는 배젖, 두 번째 수정 이벤트에서 파생 된 영양 조직으로 둘러싸여 있습니다. 모두 배아와 배유가 종피의 여러 세포 층에 의해 둘러싸여 있습니다. 종합적으로 이러한 조직은 과일 내부 개발 종자를 형성한다. 따라서, 조직 및 애기 장대 배아의 세포 별 분석은 강력하게 자신의 어려움으로 인해 손상됩니다. 그럼에도 불구하고, 늦은 구형 또는 이후 단계에서 배아는 비교적 잘 입체 현미경 하에서 미세 텅스텐 바늘을 사용하여 수동으로 해부 의무, 또는 그들을 추출하는 집게를 사용하여 초기에 약간의 압력을 적용하여 있습니다. 이러한 기술은 성공적으로 사체 또는 마이크로 어레이 하이브리드, 바이 설 파이트 시퀀싱, 또는 RNA 시퀀싱 (예 1-3)으로 epigenome 프로파일 분석에 사용 하였다. 반면, 초기 구상 단계에 수정란에서 배아의 연구는 기술적으로 도전 남아있다. 지금까지 몇 연구 담당자가고정 씨앗 섹션 4 좋은 도구 5를 사용하여 종자 내에서 개별 배아의 수동 추출에서 배아 조직 중 하나 레이저 캡처 microdissection을 (LCM)를 사용하여 젊은 배아 orted 사체 분석. 그러나 LCM 장비 초기 단계에서 일반적으로 사용되는 수동 배아 추출되지 않습니다 것은 시간이 쉽게 양도 할 수 없습니다 뛰어난 절개 기술을 소모하고 필요로하는 것입니다. 게놈 넓은 분석뿐만 아니라, 현장에서 유전자 발현 분석도 애기 장대의 젊은, 전체 마운트의 배아에서 수행하기가 어렵습니다. 어느 정도 젊은 배아는 씨앗 부드러운 압력에 의해 현미경 슬라이드에 해제 할 수 있습니다 (예를 들어 6,7 참조) 면역 염색하여 리포터 유전자 분석 또는 단백질 검출에 사용됩니다. 이 기술은, 그러나, 따라서 정량 분석​​을 방해, 높은 처리량 배아 분리를 허용하지 않습니다. 그러므로, 우리는 효율적이고 빠른 프로토콜을 개발쉽게 양도 및 다운 스트림 응용 프로그램의 다양한 적합한 설정이 간단하고 애기 장대 씨앗에서 초기 배아 분리합니다. 적절한 분리 버퍼에 플라스틱 유봉을 사용하여 에펜 도르프 튜브에 젊은 siliques에서 해부 - 기본 원칙은 부드럽게 씨앗을 분쇄하는 것입니다. 종자 추출물은 다 잘 슬라이드에 방울에 배치되어 거꾸로 현미경을 사용하여 원하는 단계에서 출시 된 배아의 존재 여부를 검사합니다. 배아는 유리 microinjector 또는 표준 실험실 피펫에 부착 microcapillary 사용하여 수집됩니다. 분자 응용 프로그램의 경우, 배아는 최소한의 볼륨에서 대상 버퍼로 전송하기 전에 새로운 절연 버퍼의 방울에 반복 릴리스 회 세척한다. 세포 학적 응용 프로그램 (기자 분석, 면역 염색, FISH)의 경우, 세척 단계는 생략 할 수 있습니다. 방법은 여러 가지 장점을 제공합니다 : (i)는 세포 학적 응용을위한 45 분 ~에 25-40 배아를 산출분자 응용 프로그램 (세척 단계 포함) lications 3-4 시간에서, (II) 그것은 특정 배아 단계의 분리는, (III)는 (IV), 쉽게 단순 설정에 따라 다른 사람과 실험실에 양도 할 수 있습니다 , 업그레이드에 대한 의무가 기본 설정을위한 저렴한 장비를 필요로하고, (v)이 성공적으로 RNA 염기 서열 8, 유전자의 특정 DNA 메틸화 분석 9, 기자 분석 (10 Raissig 등. 등 다양한 다운 스트림 응용 프로그램에 사용 된 의 준비.) 및 FISH (J. Jaenisch, U. Grossniklaus, C. Baroux되지 않은,)는 그림 5를 참조하십시오.

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Sigmacote</td>
 <td>SIGMA </td>
 <td>SL2-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse OUT</td>
 <td>Invitrogen (life technologies)</td>
 <td>10777-019</td>
 <td></td>
 </tr>
 <tr>
 <td>First- strand buffer</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen (life technologies)</td>
 <td>18064-022</td>
 <td>contained in Superscript II package</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA) 100x =10 mg/ml </td>
 <td>New England Biolabs Inc.</td>
 <td></td>
 <td>Different suppliers will also work </td>
 </tr>
 <tr>
 <td>Thin wall Capillaries 1.0 mm</td>
 <td>World Precision Instruments</td>
 <td>TW100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA LoBind tube 0.5 ml</td>
 <td>Vaudaux-Eppendorf</td>
 <td>0030108.035</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics&Delta; 30 &mu;m</td>
 <td>PARTEC</td>
 <td>04-0042-2316</td>
 <td></td>
 </tr>
 <tr>
 <td>5wells 10 mm diameter slides</td>
 <td>Electron Microscopy Sciences</td>
 <td>63421-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Formaldehyde Solution</td>
 <td>Sigma-Aldrich</td>
 <td>F1635</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost Plus slide</td>
 <td>Thermo Fisher</td>
 <td>J1800AMNZ</td>
 <td>Menzel-Gl&auml;ser</td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Amaresco</td>
 <td>0497</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Applichem</td>
 <td>A2937</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycin</td>
 <td>Fluka</td>
 <td>50050</td>
 <td></td>
 </tr>
 <tr>
 <td>SDS pellets</td>
 <td>Roth</td>
 <td>CN30.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Pestle</td>
 <td>VWR</td>
 <td>431-0094</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfine insulin syringes</td>
 <td>BD</td>
 <td>U-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC</td>
 <td>Sigma-Aldrich</td>
 <td>D5758</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Schaurlau</td>
 <td>ET00102500</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps N5</td>
 <td>Dumont</td>
 <td>0108-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer Pico Chip</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td>Nikon TMS (Japan),</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Leitz</td>
 <td></td>
 <td>Leica</td>
 </tr>
 <tr>
 <td>Micomanipulator Post mount LH1 probe</td>
 <td>Leica microsystems</td>
 <td>39430101</td>
 <td>Different brand will also do the work</td>
 </tr>
 <tr>
 <td>Vertical filament puller</td>
 <td>Sutter instrument</td>
 <td>P-20 model</td>
 <td>Other model are also suitable </td>
 </tr>
 <tr>
 <td>Cell Tram vario</td>
 <td>Vaudaux-Eppendorf</td>
 <td>5176.000.033</td>
 <td></td>
 </tr>
 <tr>
 <td>Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td>2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit Fluorometer</td>
 <td>Invitrogen (life technologies</td>
 <td></td>
 <td></td>
 </tr>
	 </tbody>
 </table>

efficient and rapid isolation of early-stage embryos from arabidopsis thaliana seeds

배유 - - 임산부 씨 integuments (또는 종피)에 의해 둘러싸여 현화 식물에서 배아 영양 조직 내에서 개발하고 있습니다. 결과적으로, 초기 단계 (구상 단계 1 셀)에서 식물의 배아의 분리가 기술적으로 상대적인 어려움으로 인해 도전합니다. 초기 단계에서 효율적인 수동 박리 강력 젊은 애기 씨의 작은 크기와 주변 조직에 배아의 부착에 의해 손상된다. 여기, 우리는 다운 스트림 응용 프로그램에 따라 1 시간에서 4 시간 40 배아까지 항복, 젊은 애기 태아의 효율적인 분리를 허용하는 방법을 설명합니다. 배아는 약간 에펜 도르프 튜브에 플라스틱와 유 봉, 250-750 씨앗을 분쇄하여 분리 버퍼에 해제됩니다. 유리는 표준 실험실 피 펫 중 하나에 연결 microcapillary (고무 튜브를 통해) 또는 유압 제어 microinjector은 방울 곳에서 배아를 수집하는 데 사용됩니다거꾸로 광학 현미경에 다 잘 슬라이드 라. 필요한 전문 기술은 간단하고 쉽게 양도 할 수 있으며, 기본 설정 값 비싼 장비를 필요로하지 않습니다. 수집 된 배아는 RT-PCR, RNA 서열, DNA 메틸화 분석, 현장 하이브리드 화 (물고기)의 형광 면역 염색 및 리포터 유전자 분석과 같은 다운 스트림 응용 프로그램의 다양한 적합합니다.

세척이 분자 응용 프로그램에 대한 예를 들어, 수행하는 경우에 우리의 배아 분리 절차 (그림 1) 4 시간 40 배아까지의 분리를 허용하거나, 시간 미만의 세척은 생략 세포 학적 응용 프로그램의 예 경우. 그림 2는 표시 높고 낮은 품질 microcapillary 팁과 그림 3은 배아 분리 시스템의 설정을 보여줍니다. 그림 4는 거꾸로 현미경에서 배아 ?...

Watch this Scientific Journal Video about Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds at JoVE.com

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

우리의 개발 초기 단계에서 배아를 분리하는 효율적이고 간단한 방법을보고<em&gt; 애기 장대</em&gt; 씨앗. 최대 40 배아 다운 스트림 응용 프로그램에 따라 1 시간에서 4 시간으로 격리 할 수​​ 있습니다. 절차는 DNA 메틸화, 리포터 유전자 발현, 면역 염색 및 형광 사체에 적합합니다<em&gt; 현장에서</em&gt; 교잡 분석.

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 ...

efficient-rapid-isolation-early-stage-embryos-from-arabidopsis

Research

JoVE Journal

Biology

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

17.7K 조회수.  University of Zürich. 우리의 개발 초기 단계에서 배아를 분리하는 효율적이고 간단한 방법을보고 애기 장대 씨앗. 최대 40 배아 다운 스트림 응용 프로그램에 따라 1 시간에서 4 시간으로 격리 할 수​​ 있습니다. 절차는 DNA 메틸화, 리포터 유전자 발현, 면역 염색 및 형광 사체에 적합합니다 현장에서 교잡 분석.

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

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

초기 치킨 태아의 성장 팩터 - 코팅 비즈 배치

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

연구

생물학

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

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

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

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 ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

씬 크로마 토그래피 (TLC)에 의해 Arabidopsis thaliana 폴라 Glycerolipid의 프로파일은 기체 - 액체 크로마 토그래피 (GLC)와 결합

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin의 분리 및 정제에서 Drosophila 태아

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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 ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

애기 씨의 높은 처리 살 균에 대 한 표준화 된 방법

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

에서 초기 단계의 배아의 효율적이고 신속한 분리

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