我们非常感谢美国俄亥俄州立大学综合癌症中心为这项工作提供资金。

1。综合复合基板的细胞系<ol><li> PDR - GFP（质粒7 M. Jasin，纪念斯隆-凯特琳癌症中心的礼物）的HeLa细胞稳定转化，在转化的选择嘌呤维持。 </li><li>细胞传代的DMEM / FB的，含10％小牛血清，GlutaMAX 2毫米，1毫米丙酮酸钠，青霉素/链霉素（100 U / mL/0.1毫克/毫升），和1.5微克/毫升的嘌呤。 </li></ol>在PDR - GFP质粒，并在稳定转化的重组基板，包含两个等位基因GFP（图1）处于非活动状态。一个等位基因是无效的，由于其编码序列的18 bp的限制性内切酶识别位点我SCEI列入。在这些细胞中的表达我的SCEI创建一个双链DNA断裂（DSB）。有这种DNA无效的GFP的第二个等位基因，但我SCEI网站上的第一个等位基因相关的部分，其序列与野生型。这第二次绿色荧光蛋白基因可作为一个序列同源重组修复的DSB捐助，将在一个活跃的绿色荧光蛋白基因，这是很容易，流式细胞仪检测结果。另外，DSB通过非同源末端连接，这将破坏我SCEI限制网站，并会导致GFP阴性细胞可以修复。在细胞同源重组的高低是由计数GFP阳性细胞的百分比。 pHeLa - DR13 - 9，该细胞系，被选为各克隆零背景GFP信号和GFP阳性细胞表达质粒，pCBASce后转与I - SCEI的比例相对较高。 （pCBASce和它的矢量控制，pCAGGS，同时提供由M. Jasin的纪念斯隆 - 凯特琳癌症中心。） 应要求作者提供的HeLa - DR13 - 9细胞株。 2。转染消耗内源性BRCA1基因突变的BRCA1加回。 <ol><li>第一染（通常是一个星期二）前一天：开始的HeLa - DR13 - 9细胞至少有90％的融合与一个10厘米的菜。 </li><li> Trypsinize 1 mL和停止反应，加入9毫升的DMEM / FB的吹打混合以及。 </li><li>添加40μL胰酶消化细胞，每孔0.5毫升/ FB的二甲醚在24孔板。 </li><li>第1天转染（星期三）：细胞应约40-50％的汇合，如果要少得多重新来过。混合转染：5 pmol的siRNA + 0.3微克BRCA1基因突变表达质粒12.5μLOPTI - MEM DNA和0.5μL脂质体2000年以12.5μLOPTI - MEM。根据脂质体协议（Invitrogen公司）进行转染。 </li><li>媒体4-6小时后更换。我们使用消耗BRCA1基因的siRNA是基于指定BRCA1基因3&#39; - UTR序列的80780到80798（登录号AY273801）序列GCUCCUCUCACUCUUCAGU。 </li><li>第2天：从24孔板trypsinizing到6孔板细胞转移。 </li><li>第3天转染：25 pmol的siRNA + 0.75微克BRCA1基因突变表达质粒DNA + 0.75微克pCBASce 62.5μLOPTI - MEM和2.5μL脂质体2000年62.5μLOPTI - MEM（或pCAGGS矢量控制）。媒体4-6小时后更换。 </li></ol> 3。分析转染。 <ol><li>第6天，trypsinize每口井的细胞，并停止在1毫升的DMEM / FB的。分析GFP表达的细胞可以在5或7天，但我们发现，每天6效果最好。 </li><li>流式细胞仪分析10000个细胞。我们使用一个碧迪FACSCalibur仪器，在美国俄亥俄州立大学综合癌症中心的分析流式细胞仪共享资源。单，活细胞的门使用的前部和侧散射参数。使用GFP荧光检测器（FL1）检测到GFP阳性细胞，结果作为一个直方图（图2，底部）。 </li><li> replated留在以上细胞可以通过荧光显微镜摄影，如果需要的话。 </li></ol> 4。代表性的成果： BRCA1基因调节的途径，通过同源重组 6,11双双链DNA断裂修复。那些已经发生同源重组修复细胞很容易检测到荧光显微镜（图2，顶部）。减少在检测到GFP阳性细胞（图2右）在BRCA1基因的结果枯竭。从FACSCalibur的输出是在X轴和Y轴（图2，底部）的细胞数，每单元的强度与GFP的直方图。在一个典型的设置从单一的实验结果，15.5％的细胞GFP阳性后，我SCEI表达和控制RNAi的枯竭（图3）。相比之下，BRCA1和矢量枯竭加回减少GFP转换至0.7％。反加野生型BRCA1或完全恢复的BRCA1基因（I21V）同源重组，获得15-16％GFP阳性细胞的检测。反加的BRCA1供应量（M18T）突变体（图3，泳道5）矢量控制添加回类似的效果。由于这种变异9作为内源性蛋白表达水平相似，因此，我们解释的BRCA1（M18T）变种非同源重组功能。 我们通常有10-20％的细胞转换我SCEI表达质粒转染后GFP阳性。重复性的BRCA1基因的消耗减少约8-10倍的GFP阳性细胞的转化。虽然GFP阳性细胞的实际比例可能会改变在一项实验中，相对于控制枯竭的BRCA1基因枯竭实验，实验中，GFP阳性细胞的比例是相当一致的。我们通过设置不羁的转换率100％，与BRCA1基因枯竭和百分比的附加回的表达结果正常化的结果。这样，我们可以结合多个实验，实验的变化是不高的。 添加BRCA1基因的突变体，迄今已取得了强劲的结果：每个BRCA1基因变种完全恢复同源重组活动，或有没有效果。在取得这些成果的一个重要的考虑是，我们的转染条件BRCA1蛋白的生产水平，约等于在未经处理的细胞内源性蛋白的浓度。太多的甚至是BRCA1基因的突变体的过度表达可能导致了一些积极的同源重组活动。 我们需要克服的一个问题是转染的毒性。 BRCA1基因本身的消耗，可以减缓细胞的生长，脂质体2000年和质粒DNA盘上的细胞数量相对的平衡，必须在一致的水平。过多的脂质体或质粒DNA会减少细胞的数量，以及细胞的数量将不足以获得可靠的流式细胞仪检测结果。与转染细胞的数量的质粒和脂质体转染试剂的平衡是很重要的，对维持细胞的可行性。 <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="图1" /> 图1。重组基板。 M. Jasin 7,12组已制定的策略是描绘。 PDR - GFP质粒包含两个绿色荧光蛋白基因有缺陷的等位基因，其中包含一个I - SCEI限制性内切酶位点。一个HeLa细胞源性细胞株包含此集成在基因组中的9的单个站点中的DNA底物，并积极修复的同源重组中的一个等位基因的转换GFP阳性的结果在我的SCEI网站的双链DNA打破。 <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="图2" /> 图2荧光显微镜检测GFP阳性细胞。细胞按照此协议，测​​试和控制亏损的细胞活跃在同源重组中，由高比例的细胞与绿色荧光蛋白表达（左）检测。耗尽的BRCA1基因的GFP阳性细胞（右）的数量在急剧下降的RNAi结果。 <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="图3" /> 图3。BRCA1基因的错义突变体的添加回结果。从单一的实验结果显示在此柱状图。在没有表达质粒转我的SCEI（巷1）有没有检测GFP阳性细胞。泳道2-6的结果都是从我SCEI表达质粒转染第3天的细胞。在细胞被耗尽无关（荧光素酶）基因和载体添加回，15.5％的细胞GFP阳性（2巷）。 BRCA1和矢量的消耗反加揭示损失的BRCA1基因同源重组的效果（泳道3）。 BRCA1和枯竭（4车道）的野生型BRCA1基因，BRCA1基因（M18T）（5巷）和BRCA1（I21V）（6车道）加回的影响。这些结果是从单一的实验中，将与至少两个以上的重复实验，以获得支持一个给定的突变BRCA1蛋白的同源重组的措施相结合。

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <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+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. 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+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

这种方法的优点是，我们可以通过同源重组使用野生型的内源性表达BRCA1基因的细胞，具有调节DNA修复的BRCA1蛋白的功能研究。我们已经采取了两个独立的研究同源重组过程中，利用RNAi介导的枯竭，获得的新方法检测DNA修复功能的BRCA1基因变体的方法。这种方法成功的关键是建立一个现成的转染的细胞株，如HeLa细胞，在基因组中，我们获得非常高的水平GFP的转换与重组基板。我们已经筛选多个克隆原来的转型，以便获得一个没有检测到的背景GFP信号，但我SCEI表达质粒转染后GFP的转换的一个非常高的水平。 使用这种方法，我们正在调查其他具体BRCA1基因同源重组功能的氨基酸残基。随着这项工作的生物见解，这些结论可以应用于临床癌症遗传学。一个高比例的BRCA1基因的错义突变的临床意义不明，因为有许多罕见的变异，其中许多是有足够的信息来确定遗传分离分析3,10,13,14癌症协会。使用这种方法，可以用来上的BRCA1基因的生物学功能调节同源重组的具体变体的后果告知妇女携带自己的基因组，这些变种之一。

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<td>11668</td>
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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

错义突变功能分析，可以是复杂的的内源性蛋白在细胞的存在。 BRCA1基因的结构与功能分析复杂，缺乏一个强大的检测全长BRCA1蛋白和细胞株，表达hypomorphic BRCA1基因蛋白1,2,3,4,5所固有的困难。我们开发出一种系统，即BRCA1蛋白在细胞内源性急性耗尽通过RNAi的目标的BRCA1基因3&#39; - UTR和共同转染质粒表达的BRCA1变异所取代。此过程的一个优点是，BRCA1和同时更换急性沉默，使细胞成长过程中没有二次突变或改编，以弥补损失的BRCA1基因的功能随着时间的推移可能出现。这种枯竭和反加的程序是在HeLa细胞衍生的细胞株同源重组活动，随时检测。同源重组的分析是基于以前发布的方法，即重组基板整合到基因组（图1） 6,7,8,9。此重组基板内处于非活动状态的绿色荧光蛋白基因的罕见切割我SCEI限制酶切位点，下游是第二个处于非活动状态的绿色荧光蛋白基因。表示在一个双链的断裂，这可能是通过同源重组修复我SCEI结果，如果同源重组修复突破，它创建了一个活跃的绿色荧光蛋白基因GFP蛋白表达的流式细胞仪检测，很容易取得的质粒​​转染。在8〜10倍，在同源重组活动的减少，导致内源性BRCA1基因的消耗和添加回野生型质粒同源重组完全恢复功能。当特定的点突变全长BRCA1基因共转染质粒表示，具体的错义突变的影响可以得分。作为一个例子，在这些细胞中的BRCA1蛋白（M18T），不明的临床意义10的一个变种，表达的表达，它未能恢复依赖BRCA1基因同源重组。通过对比，表达另一种变体，也未知意义，I21V），BRCA1基因（BRCA1基因相关的同源重组功能完全恢复。这种检测BRCA1基因的错义突变功能的策略已被应用到另一种生物系统的化验中心体的功能（凯斯卡布等，未发表意见）。总体而言，这种方法适合在任何必须分析隐性基因的错义突变分析。

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

我们提供了一个用于测试在一个组织文化同源重组修复DNA损伤的检测消耗使用RNA干扰细胞内源性BRCA1蛋白和替换它与BRCA1基因点突变，其中包含一个编码变化的BRCA1基因变体的方法。

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

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14.5K 次观看.  The Ohio State University. 我们提供了一个用于测试在一个组织文化同源重组修复DNA损伤的检测消耗使用RNA干扰细胞内源性BRCA1蛋白和替换它与BRCA1基因点突变，其中包含一个编码变化的BRCA1基因变体的方法。

视频: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

科研

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

测定野生型肝脏和牛奶样品中的唾液酸<em&gt; CMAH</em&gt;敲出小鼠

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...