This work was supported by a grant to MEM from the Dept. of Life Sciences and Systems Biology of the University of Turin, Italy. The authors thank G. Gnavi for technical assistance during some experiments. CTR is funded by the Wellcome Trust and the Royal Society (Grant Number 098436/Z/12/Z).

1.设置三轴线圈的 注 ：图1示出了用来反转GMF三轴线圈。 <ol><li>打开三轴磁力计，其探头插入三轴线圈。 </li><li>打开电脑并启动磁力仪软件，它允许数据从三轴磁力计采集。 </li><li>使用所报告的磁力计来计算GMF的量值的分量值。例如，对于磁力计的值：Bx的= 6.39μT，通过= 36.08μT，BZ = 20.40μT计算的41.94μT的场强通过使用以下方程：B = GMF + B 附加 ，其中B 额外 =（Bx的2 +由2 + BZ 2）1/2（即，41.9μT中的例子中） </li><li>打开三个直流电源（双范围：0-8V / 5A和0-20V / 2.5A，50W），每一个连接到三个couplHelmholtz线圈ES和经由一个GPIB连接（图1B）连接到计算机。 </li><li>电源的组电压，以产生所需的磁场与反相磁场矢量。例如，对于乙GMF如在步骤1.3和此处所述的仪器的线圈尺寸，设定电压为V X = 0.00，V Y = 30.52，V Z = 0.00 V，以产生一个新的所得乙=乙GMF + B 三轴线圈 =（6.38，-36.08，20.39）μT。 即，一个新的领域的大小相同乙GMF而是指向不同的方向。 </li><li>通过使用1.3中描述的步骤，检查新的领域与磁力计的软件。 </li><li>暴露植物正常和反转基因食品的条件用培养皿中第2节。 </li><li>通过保持字段等于的幅度进行假暴露实验|乙GMF |守信字段等于基因改造食品，但垂直分量改变所述场的水平分量的方向（即“北，东或西”）与相对于磁场反向条件在三轴线圈的电流相等。通过改变线圈的电压如1.5中所述要这样做。 注意：此假曝光排除了潜在的微妙的加热或从任一线圈本身或从用于控制所述线圈的电子振动的影响。 </li><li>通过施加从人员盲现场条件进行实验的其余部分和/或解释的数据运行双盲实验。 </li></ol> 2.选煤厂材料和条件对植物生长的<ol><li>使用拟南芥的种子，生态型哥伦比亚0（西0），将接成1.5毫升管和表面进行处理消毒用5％（重量/体积）次氯酸钙溶液和0.02％（体积/体积）的Triton X-100在80％的乙醇（EtOH），10-12分钟，在25-28℃下，连续振荡。然后用80％的乙醇冲洗两次，洗涤用100％的乙醇，最后用无菌蒸馏水冲洗。 </li><li>制备1升Murashige和Skoog 22（MS）的改良培养基中加入：2.297克的MS（0.5×MS基础盐混合物），10克蔗糖，去离子水至1升，pH值5.8-6.0用KOH调节。将16克琼脂和高压釜20分钟，120℃。 </li><li>凝固前，倒80毫升培养基到每个（120×120 平方毫米）正方形培养皿。播种30无菌的种子上板，然后封板与蜡状膜。 </li><li>在黑暗水平Vernalize板在4℃下2天以增强和同步发芽，然后暴露培养皿为正常或颠倒GMF。 </li><li>在内外的三轴线圈和三轴线圈外侧平行实验的竖直位置暴露种子在气候受控环境在22℃下在8小时黑暗，16小时光照光周期的时间表，使用高压钠灯（220×10 -6ê米 -2 秒 -1）。使用蓝色明胶薄膜为聚光灯以减少灯的红色分量。 </li><li> RNA提取到正常（对照）和反转（治疗）GMF条件之前暴露10天的植物。 </li><li>曝光后，利用培养皿图片。 </li><li>使用ImageJ的软件计算根长和叶面积。 <ol><li>简言之，测量培养皿的一侧，然后打开培养皿，并通过使用“直”线选项画一条线，恰好穿过板侧的图像。 </li><li>在“分析”菜单中选择“设置规模”，并插入实际距离的“已知距离”框（ 如 120毫米），然后插入的长度（mm）的单位;最后单击“全局”选项，以使可用于所有的测量设置。 </li><li>对于袋鼠长度T，使用手绘工具认真遵循根的形状。通过使用“分析”菜单中的“测量”选项测量长度。继续测量画面中的所有根和保存用于进一步的统计学分析该文件。 </li><li>对于叶面积，从“图像”菜单中的调节选项，然后在“颜色阈值”。选择单个叶片，并在“分析”菜单中选择“分析粒子”。保存用于统计分析的单独的测量。 </li></ol></li></ol> 3. 拟南芥总RNA提取，实时定量PCR（定量PCR）反应条件和引物对拟南芥 <ol><li>分别收集30芽和根30，并立即在液氮中冷冻。然后研磨在液氮与研钵和杵。 </li><li>用纯化试剂盒和RNA酶的DNA酶处理试剂盒通过厂家专业提取总RNA呃的说明。 </li><li>检查样品的质量和数量通过根据制造商的说明使用RNA纳米试剂盒和毛细管凝胶电泳。确认RNA的定量分光光度。 </li><li>用2微克总RNA，并用cDNA的逆转录试剂盒根据制造商的建议，以获得第一链cDNA随机引物的。 </li><li>使用SYBR Green I的使用ROX作为内部装载标准进行实时系统完成所有实验。 </li><li>执行与25μl的混合物组成的12.5微升2×SYBR绿定量PCR主混合物，0.5微升的cDNA和100​​nM的引物的反应。使用表 1中所列的引物。包含在控制非实时控制（使用总RNA未经逆转录以监视基因组DNA污染）和非模板对照（水代替模板）。 </li><li>计算底漆效率使用标准的所有引物对曲线法23。 </li><li>使用以下PCR条件：CRU3，COTP1，RRTF1。在95℃下10分钟，在95℃，30秒58℃，和30秒72℃40个循环的15秒; UBP6，eEF1Balpha2，ACT1，GAPC2，CAT3，TAPX，APX1，RbohD，FeSOD1 10分钟，在95℃，在95℃，20秒在57℃，和30秒72℃40个循环的15秒。 </li><li>阅读的荧光在每次退火和延伸阶段。对于所有的运行，通过在热曲线的解离段执行从55到95℃的解链曲线分析。使用解离曲线屏幕，通过成果标签访问以查看的解离曲线（荧光作为温度的函数的曲线图）。确保该实验的离解段期间收集数据集被选择用于分析使用Analysis /设定屏幕。 </li><li>确定第l通过利用基因由三个独立的RNA提取三个技术复制24,25分析了十倍系列稀释液（1至10 3倍 ）inear范围模板浓度阈值周期（Ct值）的。 </li><li>分析所有扩增曲线的实时PCR仪软件获得Ct值。校准和规范相对RNA水平与最佳的看家基因如下级别：3.11.1）通过结果选项卡进入扩增曲线屏幕上，选择斜坡或平台的数据应该使用Analysis Selection / Setup界面进行分析，然后从命令面板上的荧光菜单选择DRN（基线校正标准化荧光）。进入板样品值筛选通过的结果选项卡，显示Ct值的采样井。 </li><li>使用四个不同的参考基因[例如，细胞质甘油醛-3-磷酸脱氢酶，（GAPC2），泛素specif集成电路蛋白酶6（UBP6），Actin1（ACT1）和延伸因子1B的α-亚基2（eEF1Balpha2）]正常化的实时PCR的结果。选择顶部使用分析软件26排基因;并使用最稳定的基因进行标准化。 <ol><li>简言之，将组织在Excel表单与含有含样本名基因名与第一行第一列中的输入数据。然后从菜单栏上的分析软件。使用对话框中选择输入数据。 </li><li>接下来，检查领域的样品名称，基因名称和简单的输出而已。点击开始按钮来进行分析。选择排名最高的基因（具有最小稳定性的值），为候选基因最稳定表达。 </li></ol></li><li>绘图数据通过显示在这两个芽和根的差异倍数变化的表达。 </li></ol> 4.统计分析<ol><li> Express数据为平均值±标准差。比较合作ntrol和治疗组通过进行方差分析和Tukey检验和Bonferroni和邓恩-Sidak调整的概率试验（0.95％置信度）的分析。 </li></ol>

<ol>
	<li>Maffei, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+field+effects+on+plant+growth,+development,+and+evolution.">Magnetic field effects on plant growth, development, and evolution.</a> Front. Plant Sci. 5, (2014).</li><li>Occhipinti, A., De Santis, A., Maffei, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoreception:+an+unavoidable+step+for+plant+evolution.">Magnetoreception: an unavoidable step for plant evolution.</a> Trends Plant Sci. 19 (1), 1-4 (2014).</li><li>Harris, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+magnetic+fields+on+cryptochrome-dependent+responses+in+Arabidopsis+thaliana.">Effect of magnetic fields on cryptochrome-dependent responses in Arabidopsis thaliana.</a> J. Royal Soc. Interf. 6 (41), 1193-1205 (2009).</li><li>Phirke, P. S., Kubde, A. B., Umbarkar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+magnetic+field+on+plant+growth.">The influence of magnetic field on plant growth.</a> Seed Sci. Technol. 24 (2), 375-392 (1996).</li><li>Abe, K., Fujii, N., Mogi, I., Motokawa, M., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+high+magnetic+field+on+plant.">Effect of a high magnetic field on plant.</a> Biol. Sci. Space. 11, 240-247 (1997).</li><li>Volpe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+zero-frequency+and+oscillating+magnetic+fields+with+biostructures+and+biosystems.">Interactions of zero-frequency and oscillating magnetic fields with biostructures and biosystems.</a> Photochem. Photobiol. Sci. 2 (6), 637-648 (2003).</li><li>Belyavskaya, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+effects+due+to+weak+magnetic+field+on+plants.">Biological effects due to weak magnetic field on plants.</a> Adv. Space. Res. 34 (7), 1566-1574 (2004).</li><li>Weber Bittl, R., S, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+radical+pairs+studied+by+time-resolved+EPR.">Transient radical pairs studied by time-resolved EPR.</a> Biochim. Biophys. Acta- Bioenerg. 1707 (1), 117-126 (2005).</li><li>Pazur Galland, P., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoreception+in+plants.">Magnetoreception in plants.</a> J. Plant Res. 118 (6), 371-389 (2005).</li><li>Minorsky, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+geomagnetic+variations+affect+plant+function.">Do geomagnetic variations affect plant function.</a> J. Atm. Solar-Terrestr. Phys. 69 (14), 1770-1774 (2007).</li><li>Burda Vanderstraeten, J., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+magnetoreception+mediate+biological+effects+of+power-frequency+magnetic+fields.">Does magnetoreception mediate biological effects of power-frequency magnetic fields.</a> Sci. Tot. Environ. 417, 299-304 (2012).</li><li>Job, C., Rajjou, L., Lovigny, Y., Belghazi, M., Job, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+protein+oxidation+in+Arabidopsis+seeds+and+during+germination.">Patterns of protein oxidation in Arabidopsis seeds and during germination.</a> Plant Physiol. 138 (2), 790-802 (2005).</li><li>Wan, L. L., Ross, A. R. S., Yang, J. Y., Hegedus, D. D., Kermode, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+the+12+S+globulin+cruciferin+in+wild-type+and+abi1-1+mutant+Arabidopsis+thaliana+(thalecress)+seeds..">Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thalecress) seeds..</a> Biochem J. 404, 247-256 (2007).</li><li>Sancenon, V., Puig, S., Mira, H., Thiele, D. J., Penarrubia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+copper+transporter+family+in+Arabidopsis+thaliana.">Identification of a copper transporter family in Arabidopsis thaliana.</a> Plant Mol. Biol. 51 (4), 577-587 (2003).</li><li>Foyer, C. H., Karpinska, B., Krupinska, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+Whirly1+and+Redox-Responsive+Transcription+Factor+1+in+cross+tolerance+responses+in+plants:+A+hypothesis.">The functions of Whirly1 and Redox-Responsive Transcription Factor 1 in cross tolerance responses in plants: A hypothesis.</a> Philos.Trans.Royal Soc.B-Biol.Sci. 369 (1640), 20130226 (2014).</li><li>Myouga, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterocomplex+of+iron+superoxide+dismutases+defends+chloroplast+nucleoids+against+oxidative+stress+and+is+essential+for+chloroplast+development+in+Arabidopsis.">A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis.</a> Plant Cell. 20 (11), 3148-3162 (2008).</li><li>Mhamdi, A., Queval, G., Chaouch, S., Vanderauwera, S., Van Breusegem, F., Noctor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase+function+in+plants:+a+focus+on+Arabidopsis+mutants+as+stress-mimic+models.">Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models.</a> J. Exper. Bot. 61 (15), 4197-4220 (2010).</li><li>Bassham Contento, A. L., C, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+catalase-3+activity+as+a+response+to+use+of+alternative+catabolic+substrates+during+sucrose+starvation.">Increase in catalase-3 activity as a response to use of alternative catabolic substrates during sucrose starvation.</a> Plant Physiol. Biochem. 48 (4), 232-238 (2010).</li><li>Kangasjarvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+roles+for+chloroplast+stromal+and+thylakoid-bound+ascorbate+peroxidases+in+plant+stress+responses.">Diverse roles for chloroplast stromal and thylakoid-bound ascorbate peroxidases in plant stress responses.</a> Biochem. J. 412, 275-285 (2008).</li><li>Begara-Morales, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+cytosolic+ascorbate+peroxidase+(APX)+by+tyrosine+nitration+and+S-nitrosylation.">Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation.</a> J. Exper. Bot. 65 (2), 527-538 (2014).</li><li>Li, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtrbohD+and+AtrbohF+negatively+regulate+lateral+root+development+by+changing+the+localized+accumulation+of+superoxide+in+primary+roots+of+Arabidopsis.">AtrbohD and AtrbohF negatively regulate lateral root development by changing the localized accumulation of superoxide in primary roots of Arabidopsis.</a> Planta. 241 (3), 591-602 (2014).</li><li>Skoog Murashige, T., F, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol. Plant. 15, 473-497 (1962).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nuc. Acids Res. 29 (9), (2001).</li><li>Bustin, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+Guidelines:+Minimum+Information+for+Publication+of+Quantitative+Real-Time+PCR+Experiments.">The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.</a> Clin. Chem. 55 (4), 611-622 (2009).</li><li>Phillips, M. A., D'Auria, J. C., Luck, K., Gershenzon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+candidate+reference+genes+for+real-time+quantitative+PCR+of+plant+samples+using+purified+cDNA+as+template.">Evaluation of candidate reference genes for real-time quantitative PCR of plant samples using purified cDNA as template.</a> Plant Mol. Biol. Rep. 27 (3), 407-416 (2009).</li><li>Andersen, C. L., Jensen, J. L., Orntoft, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+real-time+quantitative+reverse+transcription-PCR+data:+A+model-based+variance+estimation+approach+to+identify+genes+suited+for+normalization,+applied+to+bladder+and+colon+cancer+data+sets.">Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.</a> Cancer Res. 64 (15), 5245-5250 (2004).</li><li>Tsukaya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+leaf+morphogenesis+in+dicotyledonous+plants.">Developmental genetics of leaf morphogenesis in dicotyledonous plants.</a> J. Plant Res. 108 (1092), 407-416 (1995).</li><li>Szymanowska-Pulka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+matters:+morphological+aspects+of+lateral+root+development.">Form matters: morphological aspects of lateral root development.</a> Ann. Bot. 112 (9), 1643-1654 (2013).</li><li>Black Bewley, J. D., Seeds, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiology of development and germination. , (1994).</li><li>Kato-Noguchi, H., Ota, K., Kujime, H., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+momilactone+on+the+protein+expression+in+Arabidopsis+germination.">Effects of momilactone on the protein expression in Arabidopsis germination.</a> Weed Biol. Manage. 13 (1), (2013).</li><li>Khandelwal, A., Elvitigala, T., Ghosh, B., Quatrano, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+transcriptome+reveals+control+circuits+regulating+redox+homeostasis+and+the+role+of+an+AP2+transcription+factor.">Arabidopsis transcriptome reveals control circuits regulating redox homeostasis and the role of an AP2 transcription factor.</a> Plant Physiol. 148 (4), 2050-2058 (2008).</li><li>Haddad, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-sensing+mechanisms+and+the+regulation+of+redox-responsive+transcription+factors+in+development+and+pathophysiology.">Oxygen-sensing mechanisms and the regulation of redox-responsive transcription factors in development and pathophysiology.</a> Respirat. Res. 3 (1), (2002).</li></ol>

The authors have nothing to disclose.

我们最近发现，转基因食品的逆转，当大多数家族被子植物谱系转移发生2时之间存在惊人的相关性。然而，尽管刺激假设和研究上的变化的基因改造食品强度效果的过多，假设GMF逆转自身可诱导在植物基因表达和形态学显著变化从未被证实。这里，我们表明，第一次，使用三轴八角亥姆霍兹线圈扭转GMF在我们的实验室，并且环境磁场的反转会导致表型变化和基因表达的植物中调制的方法。 为了获得GMF反转（或修改）在足够体积的植物生长试验（2×2×2米3），我们建立了一个八角亥姆霍兹线圈系统。该系统是不可商购（通常亥姆霍兹线圈环形和更小）和建设的费用是相当大的。重要的是，该系统提供了强大的现场修改，具有优异的时间稳定性和均匀性在修改后的磁场。 该系统的设计和建造，以减少GMF的值的正常条件千分之一或扭转任何磁场的三个方面的。然而，线圈的设计不允许以产生高磁场强度。因此，该仪器在本形式不适合用来评估的高磁场强度对植物或其它生物的影响的实验。 在实验室中，改变的GMF类似于在此方法中所描述的已用不同的方法获得，包括屏蔽由铁磁金属板具有高导磁率，其中偏离磁场和金属本身内集中他们周围的实验区。钍Ë优点使用亥姆霍兹线圈的是，该系统允许植物暴露于更多的自然条件下（光，空气流通等 ），从而使其不仅适合在体外研究 （与使用的培养皿），但也用于体内植物生长和发育的实验。我们的系统的尺寸创造一个空间，它允许抑制到天然基因食品&lt;1 / 1,000整个25×25×25厘米3的球形体积（ 见图 1A），从而使托管多个佩特里细菌培养皿或一些小盆植物生长。 这里介绍的方法已被应用到植物生物学研究;然而，该系统允许大范围的实验，包括病毒学，微生物学，以及对线虫（例如， 秀丽隐杆线虫 ），节肢动物和小动物（包括小鼠和大鼠）的研究。因此，在假设的测试，反转基因食品能以诱导形态学和转录变化也可以扩大到许多其他生命系统，可能最终甚至到人细胞。 该转基因食品是不断变化和波动。因此，在我们的实验中的一个主要挑战是提供基因改造食品的一个常数补偿，以获得所需的新基因改造食品的值。通过磁场值的连续控制，通过读取磁强计值和电压补偿时才可以实现。因此，该系统可以补偿基因改造食品的缓慢变化的一部分，但它不执行任何对于较高频率的波动。 总之，利用三轴线圈扭转GMF矢量是有助的，以证明这种逆转的GMF的载体是能够诱导植物的形态变化和差别基因表达。用该方法获得的结果提供了令人信服的证据支持的假设，即转基因食品重新versals可能是植物进化的驱动力之一在地质时间尺度2。

地球的磁场（或等价地磁场，GMF）是所有生活在地球上的生物，包括植物一个无法回避的环境因素。在转基因食品一直是地球的自然功能，因此在进化过程中，所有生物经历了行动。越来越多的证据体表明GMF是能够影响许多生物过程1。该基因改造食品是不均匀的并且有在地球的表面上在它的大小和方向显著地方的差异。在转基因食品在地球表面显示了广泛的幅度，从不到30μT几乎70μT。该转基因食品通过磁层2偏转大部分带电粒子保护地球及其生物圈来自太阳风的致命影响。 植物对环境刺激作出反应;和古典响应非生物因素如光和重力已经吨通过定义所谓的向光性和向地响应horoughly说明。很少或没有，被称为悟性和植物磁场反应的机制，尽管发表了关于这个话题，最近回顾1篇论文多如牛毛。不同的是引力场，在转基因食品植物进化过程中始终如一地改变，从而表示最近已被认为是潜在的动力，最终促进植物多样性和物种2重要的非生物胁迫因素。地磁反转（或偏移）的变化，转基因食品的极性。在地球的生命史，转基因食品的逆转发生几次。这些每一个极性转换过程中暴露出来的星球降低GMF实力周期。一些作者已经推测的低强度GMF这些过渡期间可能已经允许电离辐射从太阳风到达地球表面，从而诱导一一贯强调要活的生物体，这本来是强大到足以引起基因的改变最终导致植物进化2。 实验说明磁场对植物的影响进行详细的分析表明，大量的矛盾的报告，其特点是似是而非的生物物理相互作用机制的缺乏。许多实验都根本不现实，而另一些人缺乏检验的假设，最终都难以令人信服3。在过去几年中，在植物磁场的影响的进展和研究现状进行了审查2,4-11。近日，低和高磁场的影响进行了深入的讨论1，特别侧重于基因食品逆转事件的植物进化2的参与。 最直接的手段来证实这一假设转基因食品的逆转影响植物的进化是合成一种转基因食品逆转在实验室中通过检测植物对正常和反向的磁场条件下的反应。为了检验这一假设，我们因此建立了一个三轴亥姆霍兹八角线圈对磁场的补偿系统（三轴线圈），其能够准确地扭转正常GMF条件。 我们用拟南芥为模式植物，我们测试了逆转基因食品对一些重要基因的基因表达的影响： 十字花科3（CRU3），其编码12S种子贮藏蛋白，这种蛋白酪氨酸磷酸化和磷酸化状态被调制响应对ABA在拟南芥种子12,13; 铜运输蛋白1（COTP1），编码重金属运输/解毒家族蛋白在土壤中铜的采集和花粉发育14的主要功能;和氧化还原响应转录因子1（RRTF1）编码该ERF（乙烯反应因子）亚家族B-3的ERF / AP2转录因子家族，其中包含一个AP2结构域促进协同共激活的基因表达的途径和赋予交叉耐受性的非生物和生物胁迫15中的一员。 此外，我们还分析了五个基因参与氧化应激反应： 铁超Dismutase1，（FSD1），编码胞质酶酶促并迅速转换超氧阴离子（O 2 - ）和水 （H 2 O）与过氧化氢 ​​（H 2 O 2）和分子氧（O 2），16; Catalase3（CAT3），即编码和酶，其催化的H击穿2 O 2成水和氧17,18; Thylakoidal抗坏血酸过氧化物酶（TAPX），其编码叶绿体类囊体过氧化物酶能清除H 2 O的2 19; 抗坏血酸Peroxidase1（APX1），其编码胞质过氧化物能清除H 2 O的2和表示由NO-衍生分子20介导的翻译后修饰的潜在目标之一;和NADPH- 呼吸爆发氧化酶蛋白D（RbohD）编码生成O 2的酶-和调节生长，发育和应激反应在拟南芥21起着举足轻重的作用。 我们的现场逆转方法提供了第一手的证据表明转基因食品反转可诱导A的形态学和基因表达显著的变化拟南芥根与芽。这个协议提供了一种创新的方法来评估基因食品逆转对植物形态和基因表达的效果，并且可以用来评估GMF逆转对植物的行为的其他方面的潜在影响，并从而引导ロ讨论乐反转基因食品对植物进化的。

<table>
	<tbody>
		<tr>
			<td>three-axis magnetometer</td>
			<td>Bartington&#160;</td>
			<td>Mag-03MC</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>Magnetometer power supply</td>
			<td>Bartington&#160;</td>
			<td>Mag-03PSU</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>Magnetometer software</td>
			<td>Bartington&#160;</td>
			<td>Mag03DAM</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>DC power supply&#160;</td>
			<td>Agilent Technologies</td>
			<td>E3642A</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hypochlorite&#160;</td>
			<td>Sigma</td>
			<td>211389</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>X100&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sigma</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>GroLux Sodium vapor lamps&#160;</td>
			<td>OSRAM Sylvania</td>
			<td>600W</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plant RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>74903</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase&#160;</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>Mx3000P</td>
			<td>Agilent Technologies</td>
			<td>401512</td>
			<td></td>
		</tr>
		<tr>
			<td>2X MaximaTM SYBR Green qPCR Master Mix&#160;</td>
			<td>Fermentas International, Inc</td>
			<td>K0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Medium</td>
			<td>Sigma</td>
			<td>M5519&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish square (120x120)</td>
			<td>Sigma</td>
			<td>Z692344&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

geomagnetic field (gmf) and plant evolution: investigating the effects of gmf reversal on arabidopsis thaliana development and gene expression

其中最刺激的观察在植物进化是地磁场的发生（GMF）逆转（或偏移）和被子植物的辐射的瞬时之间的相关性。这导致了在基因改造食品极性的改变可能在植物进化中起作用的假说。在这里，我们描述了一个方法，通过使拟南芥人为地逆转GMF条件下检验这一假设。我们使用了三轴磁力计和收集到的数据被用于计算该转基因食品的大小。三个DC电源被连接到三个亥姆霍兹线圈对以及由计算机得到控制，以改变GMF条件。在培养皿中生长的植物被暴露在正常和反向的基因改造食品的条件。还进行深水暴露实验。在实验过程中暴露的植物进行拍照和图像进行分析，以计算​​根长和叶面积。拟南芥提取总RNA和定量分析在十字花科3（CRU3），铜转运蛋白1（COTP1），氧化还原响应转录因子1（RRTF1），铁超氧化物歧化酶1，（FSD1），Catalase3（CAT3），Thylakoidal的基因表达进行实时PCR（qPCR的） 抗坏血酸过氧化物酶（TAPX），胞质抗坏血酸Peroxidase1（APX1），和 NADPH /呼吸爆发氧化酶蛋白D（RbohD）。四个不同的参考基因进行分析，以归一化的qPCR结果。的四个基因最好选择和使用了最稳定的基因进行标准化。我们的数据显示，在第一次，使用三轴线圈扭转GMF极性具有对植物生长和基因表达显著影响。这支持了GMF反转有助于诱导的变化在植物发育的假设，可能证明较高的选择压力，最终导致为p兰特演变。

该协议的目的是提供一种方法，以评估逆转地磁场（GMF）是否可能影响植物的发育和拟南芥生态型胶原0三轴线圈的基因表达，如图1A被用来反转GMF时集与适当的驱动电压（图1B），获得如在步骤1.5在协议中所述。的三轴线圈的尺寸为〜2×2×2米3，允许与反转GMF条件托管多个培养皿足够的空间。对照培养在相同的环境条件，并在正常的基因改造食品的值。经过10天的接触正常或反转基因食品的条件下，植物的表型表现出明显的形态学改变。 如图2，对照植物（即，在正常GMF条件下生长）表明根长度与显著（唐恩-Sidak和邦费罗尼调整机会率&lt;0.001;学生的t = 10.68，自由度= 31）更高的值（29.41毫米; SEM = 1.04; N = 32），相对于暴露在逆转GMF植物（17.53毫米; SEM = 0.58; N = 36）。在GMF反转植物，芽的形态，也改变了显示单张扩张减少了开发。暴露在正常条件下植株4.95 平方毫米（SEM 0.025，N = 54），而暴露在扭转GMF条件植株显著（邓恩-Sidak和邦费罗尼调整机会率= &lt;0.001的平均叶面积;学生t = 31.32，DF = 53）较低的叶面积值（3.71毫米2; SEM = 0.032; N = 54）。因此，拟南芥暴露于反相GMF条件诱导两根长，叶面积的减少。 展叶和根系的生长依赖这两个分裂和细胞27的延伸上。因此，植物发育，生产力和综合健身依赖于一个最佳的拍摄开启和根系统架构&lt;SUP&gt; 28。暴露于扭转GMF条件的植物的减少的根长，叶的大小表示的感测系统的存在不仅能够感知变化的磁场强度，也能以变化的磁场“方向”相比，重力作出响应。基因改造食品的逆转可能影响植物生长的假说认为在我们的实验中，这表明转基因食品反转条件显著影响植物生长发育有说服力的证据。 还伴随着改变基因表达的形态学变化。中持家基因，最稳定的基因是延伸因子1B的α-亚基2。基因（CRU3，COTP1，RRTF1）的第一组出现了大幅度改变的基因中的表达（图3）。拍摄所有三种基因在植物中表达了显著升高（P &lt;0.05）约2.5倍，暴露在储备ð转基因食品的条件。 CRU3根表达上调在暴露在正常条件下基因食品植物根部，但显著（P &lt;0.05），下调逆转转基因食品的条件。相反，发现对COTP1和RRTF1，这是下调在正常条件下和上调GMF反转的存在（图3）。 十字花科（一个12秒球蛋白）是A的种子最丰富的储藏蛋白拟南芥和其他十字花科植物和被合成为在粗面内质网的前体。它被运送到蛋白质贮藏液泡13。幼苗发芽要求十字花科，它被用来作为氮气的初始来源的击穿。下调十字花科降解受损害的细胞结构或细胞成分的发展29,30降低胚胎发育。我们的研究结果表明，CRU3的上调与相关低级展叶和减小的根长，从而表明该基因在基因改造食品逆转并且其过度表达敏感可能有助于植物发展的减少。此外，转基因食品逆转诱导CRU3在根部显著下调，这与减少的根长。铜是用于在植物关键过程的重要辅助因子，但它施加时在过量的有害影响;因此，过表达铜运输损害植物的生长。 GMF逆转的效果COTP1在这两个芽和根一显著过表达，从而解释了降低植物生长。离子应力损害叶绿体代谢，这是紧密相连的细胞的氧化还原状态。在拟南芥转录因子RRTF1为相关联的能力的基因的表达很重要的调节氧化还原变化31。因此，当植物暴露于能够改变他们的生理和发育的外部刺激人的程序这一重要的转录因子的表达期望。对转基因食品的逆转诱导RRTF1在这两个芽和根一显著的过度表达，从而表明植株较高的氧化应激反应，逆转基因改造食品的条件。 有趣的结果是通过分析参与氧化应激五个基因获得。在一般情况下，提取和分析在枝条的所有基因没有显示当植物生长在正常或反向GMF条件（图4和图5）显著差异（P&gt; 0.05）。然而，显著下调总是暴露在相反的转基因食品的条件植物的根部观察。特别是，CAT3表现出最高的下调（图5），接着在由APX1，FSD1，RBOHD和TAPX（图4）顺序的下调。 交叉耐受性到生物和生物胁迫是由参与几个生化途径不同的基因的激活提供。RRTF1转录因子促进协同共激活这些途径15,31的基因表达，并且可以潜在地参与氧化应激32。因此，预计RRTF1的上调时除氧降低。根清除酶的下调相关与RRTF1的上调，其作用在响应于氧化应激增加。 CAT3，APX1和TAPX的戏剧性根下调表示根细胞以清除H 2 O的2，这是伴随着由FSD1下调到dismutate超氧阴离子的能力降低的能力降低。氧化应激反应是根，这似乎是相反的转基因食品认知的主要部位高。 图1.地磁场补偿系统。 （A）的三轴线圈（包括一对线圈八角每个三个方向轴的）用于反向地磁场矢量。 （B）的一种计算机控制的电源被连接到各对亥姆霍兹线圈。 （图中电压是任意） <a href="https://www.jove.com/files/ftp_upload/53286/53286fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53286/53286fig2.jpg" /> 图2.影响地磁逆转对拟南芥的形态 。曝光后，控制植物十天（ 即那些暴露在正常条件下GMF）显示显著更大的根长，更EXPANDED传单相比，暴露于扭转GMF条件的植物。公制吧= 18毫米。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53286/53286fig3.jpg" /> 图对拟南芥基因表达3.影响地球磁场逆转。曝光后十天，控制和处理的植物的总RNA提取和实时​​PCR表达分析来分析。反转的转基因食品的效果，诱导该测试CRU3，十字花科3所有基因的基因表达的急剧变化; COTP1，铜传输蛋白1; RRTF1，氧化还原响应转录因子1。线表示标准误差;星号表示显著（P &lt;0.05）复之间的差异暴露在扭转，转基因食品的正常条件下的蚂蚁。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53286/53286fig4.jpg" /> 图对拟南芥的抗氧化剂相关基因的表达4.影响地球磁场逆转。曝光后十天，控制和处理的植物的总RNA分离，并使用实时PCR用于基因表达分析。反转的转基因食品的作用是诱导芽基因表达无显著的变化;然而，大幅下调，观察植物的根系基因表达逆转基因食品的条件下生长TAPX，Thylakoidal抗坏血酸过氧化物 ; APX1，抗坏血酸Peroxidase1; FSD1，铁超Dismutase1; RbohD，NA。DPH /呼吸爆发氧化酶的活性蛋白D线表示标准误差;星号表示受到扭转，转基因食品的正常条件下植物之间显著（P &lt;0.05）的区别。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53286/53286fig5.jpg" /> 图5.影响地球磁场逆转对拟南芥过氧化氢 ​​酶3（CAT3）基因的表达 。曝光后十天，控制和处理的植物的总RNA分离，并使用实时PCR用于基因表达分析。反转的转基因食品的作用是诱导芽基因表达无显著的变化;然而，急剧下调中观察到颠倒GMF条件下生长的植物的根的基因表达。线表示标准埃罗- [R;星号表示受到扭转，转基因食品的正常条件下植物之间显著（P &lt;0.05）的区别。 <a href="https://www.jove.com/files/ftp_upload/53286/53286fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression at JoVE.com

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

地磁场（GMF）和植物进化：调查逆转基因食品对影响<em&gt;拟南芥</em&gt;开发与基因表达

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

geomagnetic-field-gmf-plant-evolution-investigating-effects-gmf

Research

JoVE Journal

Developmental Biology

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

13.0K 次观看.  University of Turin. This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

视频: Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...