저자는 우리 실험실과 DRS에 털이 루트 기술을 소개하는 박사 크리스 테일러 (오하이오 주립 대학)에 감사드립니다. Senthil Subramanian (사우 스다코타 주립 대학), 프로토콜 개선을위한 후안 장 (Ludong 대학 산동 중국). 우리는 또한이 비디오를 만드는 데 도움이 박사 하오 쳉 (남경 농업 대학) 감사합니다. 이 작품은 오우으로 DOE (DE - SC0001295), NSF (MCB - 0923779)과 USDA (2010-65116-20514)에서 부여에 의해 일부 지원

다음 프로토콜은 모델 콩과의 식물 종 M.에 털이 루트 복합 발전소를 생성하는 데 사용되었습니다 truncatula. 비슷한 프로토콜은 적어도 8 식물 종의 1-4에 맞게 조정되었습니다. 우리는 M. 사용 truncatula 털이 루트 복합 식물 뿌리와 결절 개발에 유전자 기능을 연구하기 위해 서요. 털이 루트 복합 발전소를 생성 2),, 3) 공생 Rhizobia 감염, 그리고 4) 유전자 변형 루트 식별을 준비하는 식물 재료에게) 1 : 프로토콜은 4 개의 섹션으로 분리되었습니다. 우리는 합성 유전자 변형 식물의 뿌리 3 검사의 기자로 녹색 형광 단백질 (GFP) 유전자를 포함하는 바이너리 벡터를 사용합니다. 항생제 기반의 선택에 비해 GFP 기반의 심사는 빠르고 쉽게, 그리고 저렴합니다. 우리 구조에서 ER 표현 최적화된 GFP 유전자는 유전자 변형이 아닌 유전자 변형 뿌리 사이에 쉽게 구별 수 있도록 유전자 변형 뿌리에 강한 제정 GFP 신호를 가지고있는 슈퍼 ubiquitin 발기인에 의해 구동됩니다. 1. 식물 재료 준비 <ol><li> M. truncautla의 씨앗은 온실 재배 식물 (50 % 상대 습도, ​​8분의 16 H 조명 / 어두운, 18분의 22 ° C 주 / 야 온도)에서 수확하고 있습니다. 성숙한 종자는 4 ° C.에 저장할 수 있습니다 </li><li> 약 100 씨앗은 정기 잡고 10 분 10 ML 집중 황산 (95-98%, 시그마 - 알드리치, MO)에 scarified 있습니다. </li><li> 씨앗은 부드러운 교반과 멸균 물을 5-7 번 씻어서 있습니다. </li><li> 씨앗은 다음 6 시간 동안 실온에서 물에 흠뻑 젖고 ° C가 36-48시간에 대한 발아를 동기화 4 보관됩니다. </li><li> 약 20-30 종자는 페트리 접시에 배치 젖은 여과지에 보급하고 있습니다. 그들은 22 ° C 유지를 μmol.m -2 8분의 16 H 조명 / 어두운주기, 그리고 40. S -1 빛의 강도는, 5-6 일간. </li><li> Germinated 모종은 흙으로 전송하고 한 달 동안 온실에 배치됩니다. </li></ol> 2. 헤어 루트 복합 식물을 생성 <ol><li> 관심 유전자를 갖고 이진 구성 요소 A.으로 변형되었다 표준 프로토콜을 사용 rhizogenes. 우리는 유전자 변형 조직 검사를 용이하게 마커로서 GFP 유전자를 포함하는 바이너리 벡터의 집합을 꾀하고있다. 이 벡터는 다양한 발기인을 포함하고 모두 이상 표현이나 타겟 유전자 3,5의 입을위한 게이트웨이 복제 사이트 (Invitrogen, 칼스 배드, CA) 있습니다. </li><li> A. rhizogenes의는 28에서 적절한 항생제 선택과 50ml LB 배지 ° 16~24시간 들면 C에서 양식입니다. </li><li> 박테리아는 질소없는 식물 영양소 솔루션 (표 1, 6) = 0.3를 수집하여 OD 600의 최종 농도에 다시 중지합니다. </li><li> 털이 루트 재생, 소독 지원 매트릭스 (이하 &quot;록 양모&quot;Hummert 국제, 지구시, MO)을 지원하기 위해하는 것은 약 3cm 3 플러그로 절단됩니다. 하나는 페트리 접시에 4 플러그를 놓으십시오. </li><li> 구멍 explants의 삽입을 용이하게하기 위해 피펫 팁을 사용하여 각 플러그 위에 찌르고있다. A. rhizogenes (5 ML)은 각 플러그인에 추가됩니다. </li><li> 2-3 겨드랑 꽃봉오리와 촬영 섹션 슬랜팅 절단에 의해 excised 수 있습니다. explant은 다음 플러그에 삽입하고, 22 ° C 3 주에 대한 성장이다. 우리는 물이 그들을 5 ML 질소 무료 솔루션을 필요한 경우와 다음, 처음 10 일 동안 물을없이 Medicago의 explants을 유지. 우리는 혀끝의 분열 조직을 제거하는 것은 우발적인 뿌리의 형성을 낮출 수있는 것으로 나타났습니다. </li></ol> 3. 공생 Rhizobia (Sinorhizobium Meliloti) 감염증 <ol><li> 3 주 후에 어떤 우발적인 뿌리가 록 양모에서 나타날 것입니다. 털이 루트 복합 식물은 록 양모에 관계없이, 모래 또는 vermiculite (소독)으로 전송됩니다. 그들은 22 온실에서 재배 ° C, 8분의 16 H 조명 / 어두운 사이클, 300 μmol.m -2. S는 1 주일 -1 빛의 강도. </li><li> rhizobia 감염, S. 이전에 meliloti 1201 50 ML 효모 추출물 - mannitol 30 ° C (표 2, 6,7)에서 7 일 동안 매체 양식입니다. </li><li> 다시 정지 rhizobia 세포는 OD 600의 최종 농도 = 0.08 사용하여 질소 무료로 영양 솔루션으로 희석하고 있습니다. S.의 10 ML 신선한 서스펜션 meliloti는 결절 형성을 유도하기 위해 각각의 합성 공장에 적용했다. </li></ol> 4. 유전자 변형 루트 식별 <ol><li> 두 주 rhizobi​​a 접종 후, 합성 식물은 물에 세척에 의해 뿌리입니다. 뿌리는 쉽게 물에 모래 또는 펄라이트 분리 수 있습니다. </li><li> 우리는 UV - 현미경 (니콘 SMZ1500, 여기 460 - 500nm, 505nm 이색성, 510nm 이상의 배리어)에서 털이 뿌리를 놓으십시오. 형질 뿌리는 GFP 양성하고 우발적인 뿌리는 GFP 아무것도 없습니다. 우리는 다음 뿌리 accordin를 수집g GFP 신호, 그리고 결절 번호를 카운트 루트 길이를 측정하고, 측면 루트 밀도를 계산합니다. GFP 부정적인 뿌리는 컨트롤로 사용됩니다. </li></ol> 5. 대표 결과 우리의 실험에서 새 머리카락 뿌리는 A. 후 2-3주에 explants에서 재생 rhizogenes innoculation. UV - 현미경, GFP 유전자를 가지고 유전자 변형 뿌리는 강한 녹색 형광 (그림 1)를 보여줍니다. 재생 뿌리와 GFP 긍정적인 뿌리 부분의 금액은 explants 및 복합 plants.On 평균의 성장 환경의 조건에 따라, 생산 뿌리의 25 %가 유전자 변형 털이 뿌리 셋 있었다. 변환 효율, 1)를 향상시키기 위해 로 비디오에 표시된 플라스틱 커버 운명은, 처음 몇 일 동안 성장 트레이에서 습도를 유지하기 위해 충분하고, 식물은 처음 며칠 동안 약간의 물을 필요로합니다. 과도한 관수는 털이 루트 형성에 해로운이며 2) Agrobacterium innoculant의 농도가 변화하는 것이 중요합니다. 세포의 과도한 금액은 털이 루트 형성 또는 결절 형성에 도움이되지 않습니다. 결절 형성은 물을 솔루션은 질소 무료 있어야합니다, 그렇지 않으면 몇 nodules가 형성됩니다. 털이 루트 복합 식물은 시작 물질로 cotyledons 또는 entact의 모종을 사용하여 생성할 수 있습니다. 위의 프로토콜만이 사소한 변경은 다른 조직 8 털이 뿌리를 생성할 수 ncessary 있습니다. 중요한 것은 각각의 털이 루트는 독립적인 변환 이벤트입니다. 따라서 하나의 합성 공장에서 관찰 phenotypes 여러 복합 식물의 반복에 의해 확인하는 데 필요한 몇 가지 transforamtion 이벤트의 총합으로 이루어진 <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="그림 1" /> 그림 1. A. 형질 전환 뿌리는 털이 뿌리에서 밖으로 정렬할 수 있습니다. B.의 Nodules는 털이 루트 합성 공장에서 형성되었다. 우리는 4 주 된 M. 배치 UV - 현미경과 GFP 긍정적인 뿌리 아래에 truncatula 털이 뿌리 식물을 쉽게 확인할 수 있습니다. (레드 애로우 : GFP 루트, 노란색 화살표 : 비 - GFP 루트) <table border="1"><tbody><tr><td> 재고 </td><td> g/200ml </td><td> ML 재고 / 리터 </td></tr><tr><td> MgSO 4 0.7 H 2 O </td><td> 12.3 </td><td> 2 </td></tr><tr><td> CaCl 2 0.2 H 2 O </td><td> 14.7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 0.3 H 2 O </td><td> 6.8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td> 철 Club 호텔 3 0.6 H 2 O </td><td> 0.49 </td><td> 2.5 </td></tr><tr><td> Micronutrients </td><td> 아래 참조 </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Micronutrients </td><td> 리터 당 g </td></tr><tr><td> H 3 BO 3 </td><td> 0.142 </td></tr><tr><td> MnSO 4. H 2 O </td><td> 0.077 </td></tr><tr><td> ZnSO 4 0.7 H 2 O </td><td> 0.1725 </td></tr><tr><td> CuSO 4 0.5 H 2 O </td><td> 0.037 </td></tr><tr><td> NaMoO 4. H 2 O </td><td> 0.024 </td></tr><tr><td> CoCl 2. H 2 O </td><td> 0.0025 </td></tr><tr><td> NiSO 4 </td><td> 0.001 </td></tr></tbody></table> 표 1. Nitogen없는 영양 솔루션 <table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0.5g </td></tr><tr><td> NaCl </td><td> 0.1g </td></tr><tr><td> MgSO 4 명이 7H 2 O </td><td> 0.2g </td></tr><tr><td> 효모 추출 </td><td> 0.4g </td></tr><tr><td> Mannitol </td><td> 10g </td></tr><tr><td> 산도 = 6.8 </td><td></td></tr></tbody></table> 표 2. 효모는 (리터 당) mannitol 미디어를 추출

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

털이 루트 합성 공장을 생성하는 것은 많은 dicot 종족에 대한 유전자 변형 물질의 다량을 얻을 수있는 빠르고 쉬운 방법입니다. 이 방법은 유전자 변형 종자를 생산할 수 없지만, 그것은 몇 주 안에 유전자 변형 물질을 생성할 수 있습니다. 방법은 어려움이 조직 문화를 구축하거나 안정적인 transformants 생성을 가지고 식물에 특히 적합합니다. 수년에 걸쳐, 우리는 유전자 기능, 발기인 기능, microRNAs, 루트 및 측면 루트 개발, 국방 및 비생물적 스트레스 반응, 유괴 및 기타 공생 프로세스, 호르몬 반응, 신진 대사 프로 파일링, 유전자 프로 파일링, proteomic 분석, 연구를이 기술을 사용한 다른 생물 학적 처리합니다. 프로토콜은 강력하고 복제할 수 있습니다.

generation of composite plants in medicago truncatula used for nodulation assays

Agrobacterium의 tumerfaciens 마찬가지로, Agrobacterium rhizogenes의는 플라스미드 (리) 루트 - 유도하는 자율을 바탕으로 식물 세포에 외국 DNAs을 전송할 수 있습니다. 답변 rhizogenes는 식물 조직에 털이 루트 형성 원인 및 변환 후 복합 발전소를 형성할 수 있습니다. 이러한 복합 식물에서 생성 뿌리의 일부가 야생 타입 T - DNA와 엔지니어링 이진 벡터를 들고, 유전자 변형되며, 쏘고는 에너지 및 성장 지원을 제공하기 위해 제공 아직되지 않은 유전자 변형되는 동안. 이러한 털이 루트 복합 식물 유전자 변형 종자를 생산하지 않지만 식물 연구에 매우 유용 이러한 합성 공장을 만드는 중요한 기능은 여러 가지가있을 수 있습니다. 첫째, 광범위한 호스트 범위, ​​A. rhizogenes는 인류의 다양한 유전 공학을 허용, 특히 많은 식물 종, dicots를 변환하실 수 있습니다. 둘째, A. rhizogenes 직접 조직 및 explants 감염, 이전에 변환 할 조직 문화는 완강히 저항하는 식물 종의 변화에 이상적, 복합 발전소를 얻을 필요가 없습니다. 또한, 유전자 변형 루트 조직은 주 만에 생성할 수 있습니다. Medicago truncatula 들어, 우리는 정상적인 꽃 물놀이 Arabidopsis 변형보다 같은 짧은 셋처럼 주 형질 뿌리를 얻을 수 있습니다. 전반적으로, 털이 루트 복합 플랜트 기술은 유전자 기능을 연구하고 관련 - phenotypes를 루트에 다목적하고 유용한 도구입니다. 여기 털이 루트 복합 식물이 어려운 변환 종 M. 공장 - rhizobium 상호 작용과 유괴를 공부하는 데 사용할 수있는 방법을 보여줍니다 truncatula.

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

털이 루트 복합 식물이 어려운 변환 종의 식물 rhizobi​​um 상호 작용과 유괴를 공부하는 데 사용할 수있는 방법을 우리가 보여<em&gt; Medicago truncatula</em&gt;.

의 복합 식물의 생성 Medicago truncatula 유괴의 Assays 사용

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

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Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

16.0K 조회수.  Donald Danforth Plant Science Center. 털이 루트 복합 식물이 어려운 변환 종의 식물 rhizobi​​um 상호 작용과 유괴를 공부하는 데 사용할 수있는 방법을 우리가 보여 Medicago truncatula.

비디오: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

의 간질 발작의 돌연변이에 대한 Neurocircuit의 Assays Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

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생물학

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

식물의 성적 증명서의 정확한 현지화에 대한 원위치 하이브리드화에서

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

의 대사 산물의 조사를위한 MALDI-질량 분석 이미징<em&gt; 개자리 truncatula</em&gt; 루트 결절

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

측정 공간적 칼슘<sup&gt; 2 +</sup애기 장대 식물에서&gt; 신호

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM 마이크로코일 성능 교정 및 사용 에 설명 된 메디카고 트런카툴라 뿌리에 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.