우리 작품은 VC에 NIH / NIGMS, NSF, USDA / NIFA 및 바드에서 기금에 의해 지원됩니다.

배경 식물 세포 연결을 통해 macromolecules의 Symplastic 교통, plasmodesmata는 많은 식물 병리학과 생물학에 관심이 있습니다. 예를 들어, 여러 바이러스 단백질은 바이러스 운동 1-3 있도록 plasmodesmal 크기 제외 한도를 규제하는 것으로 알려져 있습니다. 또한, 그들이 중요한 발달 규제 중 일부 내생 단백질은, 비 셀 자율적으로 4 기능, 아마도 plasmodesmata을 통해 세포에서 세포로 이동 간주됩니다. 따라서 식물 세포 사이의 macromolecular 운송을 파악하고 시각화하는 신뢰할 수있는 방법론이 훨씬 수요에 있습니다. 1) 대상 식물을 성장 높은 변환 효율을위한, 건강, 강력한 식물을 사용해야합니다. 1. Arabidopsis 식물 짧은 photoperiod (130-150 μE m -2 -1의 빛 8 시간 23 °와 환경 제어 챔버의 냄비에 프로 믹스 BX 한 Arabidopsis 공장 (10cm X 10cm X 10cm) 성장 C/16 20 시간 어두운 ° C)과 6~8주 5 40-65% 상대 습도. 5 설명된대로 상용 제품을 종종 그들을 비옥. 15mm X 35mm (길이 측정은 잎자루 포함)보다 큰 크기로 단풍이 실험을 위해 선택됩니다. 2. N. benthamiana와 N. tabacum 긴 photoperiod (23 ° C에서 130-150 μE m -2 -1의 빛을 16 시간과 환경 제어 챔버에 냄비 (20cm X 20cm X 20cm)에 프로 믹스 BX 한 식물을 성장 / 8 20 어두운 HR ° C) 및 7~10주에 대한 40-65% 상대 습도. 묘사와 같은 상용 제품을 종종 그들을 비옥. N. 대한 사이즈 50mm보다 큰 X 70mm와 잎 N. benthamiana에 대한, 또는 100mm X 125mm tabacum가 (이 길이 측정 잎자루에 포함되지 않습니다) 실험 선택됩니다. DNA - 코팅 골드로 진 건 카트리지 2) 준비 Microparticles 이 실험 단계에 대한 프로토콜은 이전에 여섯 자세히 설명되었습니다. 그것은 높은 농도 (~ 1 μg / μl) 실험의 나중 단계 동안 공촛점 현미경 분석의 용이성을위한 최고의 변환 효율을 얻기 위해에 잘 정화 플라스미드 DNA를 사용하는 것이 매우 중요합니다. 형광 신호 클러스터와 어울리는 세포의 숫자가 symplastic 수송의 범위 (즉,의 지표로 사용되기 때문에 분석을 위해, 그것은 준비 카트리지가 높은 주파수에서 동시에 둘 이상의 인접한 세포를 변형하지 않는 확인할하는 것이 중요합니다 , multicell 신호 클러스터)가 움직임을 나타내는 반면 신호를 포함하는 하나의 조직이 어떤 움직임을 나타냅니다 없습니다. 카트리지 각의 품질은 단백질 16-20시간 포스트 충격의 표현을 분석하여 확인하실 수 있습니다. 우리 프로토콜 6는이 실험 따라서 적당이 시간 프레임에있는 모든 표현 행사 &lt;3 %,에서 multicell 표현 클러스터를 얻을 카트리지를 생산하고 있습니다. DNA - 코팅 Microparticles 3) Biolistic 배달 <ol><li> 날카로운 면도날과 같은 발달 단계 (즉, 같은 크기와 같은 나이로, 1 단계를 참조)의 잎을 제거하고 즉시 평면 스티로폼의 표면에까지 직면하고있는 abaxial 측면으로 그들을 놓으십시오. 잎사귀의 abaxial 측면 때문에 그들의 비교적 작은 크기의 Arabidopsis 잎은, 윈도우 화면 메쉬의 조각으로 덮여 있어야합니다. 그 낮은 trichome 밀도와 얇은 표피로 인해 충격을 위해 더 나은 기판을 대표하고, 메쉬 다음 pushpins과 안전 스티로폼 표면 수 있습니다. 유지은 평면은 입자 전달의 효율성을 증가하고 microbombardment 동안 조직에 대한 손상을 최소화 떠납니다. </li><li> 총으로 DNA - 코팅 microparticles (1 단계에서 준비)와 카트리지를 삽입합니다. Arabidopsis 들어, 촬영은 0.6 - μm의의 microparticles 1 - μm의 microparticles, 그리고 140-160 PSI에 대한 80-110 PSI의 압력에서 수행됩니다. N. 들어 benthamiana 담배는 촬영은 0.6 - μm의 microparticles 1 - μm의 microparticles, 그리고 160-180 PSI에 대한 PSI의 압력 100-120에서 수행됩니다. 각 잎은 직경 10-12mm의 영역 microparticles을 확산 한방에 충분한 면적을 가지고 있기 때문에 Arabidopsis 위해, 우리는 일반적으로 잎 당 하나의 카트리지를 사용합니다. 큰 N.으로 benthamiana와 담배 잎 같은 잎의 여러 bombardments은 가능하지만, 그들은 (토론 참조) 같은 발달 단계에서 잎 영역을 대상으로 중간 리브의 양쪽에 대칭 위치에서 수행되어야합니다. </li><li> 스티로폼 표면에서 잎을 제거하고 그들에게 내가 배치서부 유럽 표준시 왓먼 필터 종이 3 레이어가 아닌 페트리 접시는 Parafilm와 페트리 접시를 밀봉하고, 그들의 단백질 제품의 배달 transgenes와 휴대 전화의 운동 잠재력의 표현을 허용하기 위해 36-48 시간을위한 실내 온도에서 품어 . </li></ol> 단백질의 표현 4) 영상 transiently 표현 태그 단백질의 형광 신호는 공촛점 현미경에 의해 시각입니다. 치료는 각 테스트 단백질의 검출을위한 최적의 현미경 설정을 찾을로 이동해야합니다. 예를 들어, 담배 모자이크 바이러스 운동 단백질 (TMV MP) 1-3,6의 plasmodesmal 지방화로 약한 신호 강도와 제한 세포내 축적을 보여 단백질은 높은 해상도와를 내실수 40X 객관적인 렌즈, 아래의 관찰해야합니다 감도는 반면, 같은 무료 YFP와 같은 강력한 신호 농도, 세포질과 유통을 보여 단백질 빠른 영상에 대한 공촛점 줌 기능이있는 객관적인 10X 렌즈 (그림 1 참조)에서 시각하실 수 있습니다. Symplastic 수송은 형광 신호를 포함하는 multicell 클러스터의 모양에서 유추됩니다. 각 클러스터의 클러스터와 같은 세포의 수를는 휴대 전화 전송의 범위 나타내는 것입니다. 신뢰할 수있는 데이터를 얻으려면, 최소한 100 표현 클러스터는 각 실험 시스템마다 기록해야합니다. 예를 들어, 단백질의 휴대 전화의 움직임이 총 200 표현 클러스터의 두 가지 다른 유전 배경 (예, 야생 종류와 유전자 변형 식물)에 비교하면이 기록되어야합니다. 중요한 것은, 실험, 서로 비교해야하는 결과가 동시에 진행되어야합니다. 5. 대표 결과 아래의 그림은 찬란 태그 단백질의 symplastic 교통 검출을위한 대표적인 실험을 보여줍니다. 패널은 A와 B는 N.의 다음 microbombardment를 획득하는 전형적인 공촛점 이미지를 표시 TMV MP - YFP - 표현과 benthamiana 리프가 건설. 패널에서 TMV MP - YFP의 A, symplastic 운동은 YFP 신호의 multicell 클러스터의 모양에 따라 관찰합니다. 모든 transiently 표현 TMV MP - YFP은 일부 microbombardments (패널 B)의 단일 세포 신호에 의해 입증 이동시킬 수 없습니다. 통계에 &lt;계산 신호 클러스터의 40 %, TMV MP - YFP는 반면, 세포 사이를 이동할 수없는&gt;의 클러스터의 60 %, 단백질은 두 세포 확산이 가장 자주 있기 때문에 2-5 셀 사이를 이동 (ca. ~ 50 % 가지 경우) (그림 1C). 타고난 운동 활동을하지 않고 비교적 작은 분자 크기의 단백질은 휴대 전화를 이동하는 PD를 통해 확산하실 수 있습니다. 예를 들어, 무료 YFP, 또는 1xYFP은 (ca. 27 KDA, 패널 D), 계산 클러스터 (패널 C)의 30 %가 여러 세포 사이에 확산됩니다. 이 이외의 특정 확산은 TMV MP - YFP 융합 단백질 (57 KDA)의 크기 비교이며, 완전히 셀 자치있는 translational YFP의 주차 또는 2xYFP (54 KDA, 패널 E)에 대한 발생하지 않습니다 (그림 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="그림 1" /> N.의 symplastic 교통 분석에서 얻은 그림 1. 전형 결과 benthamiana 잎 조직. TMV MP - YFP의 (A, B) 시각. 신호 클러스터 (C) Quantitation. 1xYFP 및 2xYFP, 무료 YFP 각각 translational YFP 이합체. 1xYFP의 (D) 시각. 2xYFP의 (E) 시각. micrographs에서, 왼쪽 패널 YFP 신호를 표시하고 오른쪽 패널 합병 YFP의 이미지 (녹색) 및 엽록체의 autofluorescence (흰색) 신호를 보여줍니다. 이미지는 단일 공촛점 섹션입니다. micrographs에 별표 YFP 신호를 보여주는 표피 세포를 보여줍니다. 바 = 50 μm의.

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment.">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat Protoc. 4, 71-77 (2009).</li><li>Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+but+not+branched,+plasmodesmata+allow+the+nonspecific+trafficking+of+proteins+in+developing+tobacco+leaves.">Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.</a> Cell. 97, 743-754 (1999).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11, 309-322 (1999).</li><li>Gisel, A., Barella, S., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+symplastic+trafficking+during+development+in+Arabidopsis+thaliana+apices.">Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.</a> Development. 126, 1879-1889 (1999).</li><li>Kim, I., Cho, E., Crawford, K. M., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+movement+of+GFP+during+embryogenesis+and+early+seedling+development+in+Arabidopsis.">Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis.</a> Proc Natl Acad Sci USA. 102, 2227-2231 (2005).</li><li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+(TMV)+replicase+and+movement+protein+function+synergistically+in+facilitating+TMV+spread+by+lateral+diffusion+in+the+plasmodesmal+desmotubule+of+Nicotiana+benthamiana.">Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.</a> Mol Plant Microbe Interact. 21, 335-345 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

symplastic 교통 분석의 성공에 대한 열쇠는 통계적으로 중요한 쉽게 detectible 신호 클러스터의 생산을 허용 높은 변환 효율을 얻을 수 있습니다. 이것은 건강, 강력한 식물에서 수확한 잎을 사용하고 순수하고 집중 DNA 준비로 코팅된 금 입자를 준비 얻을 수 있습니다. 같은 성장 단계에서 나뭇잎을 사용하면 분석의 신뢰성을 위해 매우 중요합니다. Plasmodesmal 조리개는 differentially...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
<tr>
<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
<tr>
<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
<tr>
<td>Helium gas regulator</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2413</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

여기, 우리는 planta에서 휴대 전화의 macromolecular 수송의 범위를 감지하고 평가하는 간단하고 빠른 프로토콜을 제시한다. 이 프로토콜에 관심이 찬란 태그 단백질은 transiently의 인코딩 DNA의 전달 구조 biolistic 다음과 식물 조직에 표시됩니다. 태그 단백질의 내부 및 세포 분포는 다음 공촛점 현미경으로 분석됩니다. 우리는 분석하기 위해 단계별 절차를 제공 세부 사항이 기술을 설명하는 세 식물 종 (种)의 symplastic 단백질 수송의 범위, Arabidopsis thaliana, Nicotiana benthamiana 및 N. 평가 tabacum (담배).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

식물 세포 사이 Macromolecular 거래는 transiently 관심 찬란 - 태그 단백질을 표현하고 공촛점 현미경하여 내부와 세포 분포를 분석하여 평가하실 수 있습니다.

휴대 전화의 Macromolecular 교통 분석 Planta에 Biolistic 포격을 활용

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

a-cell-to-cell-macromolecular-transport-assay-planta-utilizing

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K 조회수.  State University of New York at Stony Brook. 식물 세포 사이 Macromolecular 거래는 transiently 관심 찬란 - 태그 단백질을 표현하고 공촛점 현미경하여 내부와 세포 분포를 분석하여 평가하실 수 있습니다.

비디오: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

진 건 글머리 기호 및 슬라이스 문화 뉴런의 Biolistic Transfection의 준비

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

연구

생물학

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

다공성 매체에서 미생물 교통 유학에 대한 간단한 Microfluidic 시스템 : 미생물의 창

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

트렌스 제닉의 생성 C. 엘레간스 Biolistic 변환에 의해

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

커튼에서 Agrobacterium - 중재 바이러스 유발 유전자 입을 분석

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-의존 CFTR의 Macromolecular 신호 단지의 체외 분석

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

배양<em&gt; 예쁜 꼬마 선충</em &gt; 미세 폭격에 의한 무균 액체 미디어 및 형질 전환 웜의 생성에

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

방법은 3D로 재배 원주민 소장과 카코 2 세포에서 상피 전송 단백질의 기능과 발현을 연구하는

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

마이크로 패턴 세포에서 세포 외세포증의 주당 측량 파라미터를 정량화

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.