作者感谢加拿大自然科学和（NSERC）工程研究理事会资助这项工作。

1.媒体和文化的制备<ol><li>对于LB +放大器:制成500毫升之的Luria-BERTANI肉汤（LB）的使用在121℃高压釜中进行30分钟灭菌。补充有氨苄青霉素至4℃，得到的100微克/毫升氨苄青霉素和存储的终浓度直到需要。 </li><li>对于TB +放大器:准备大（500毫升以上）ministring生产量。制备使用在121℃高压釜中进行30分钟灭菌600毫升无菌了不起肉汤（TB）的。补充有氨苄青霉素至4℃，得到的100微克/毫升氨苄青霉素和存储的终浓度直到需要。 </li><li>对于LB琼脂+放大器:由制备添加了15克/升琼脂LB +安培准备无菌的LB琼脂平板上。等分约15 - 20ml如无菌培养皿中并储存倒挂在4℃直至需要。 </li><li>准备W3NN [PDMS]的连胜板经无菌裸奔W3NN [PDMS]在LB琼脂+放大器细胞。孵育板overni向右在30℃下，直到单个菌落可以观察到。不要孵化高于此温度W3NN避免derepressing protelomerase表达。 </li><li>无菌地转移5ml的LB +安培到无菌试管中。接种此体积与来自划线平板的单个菌落制备W3NN [PDMS]的过夜培养。孵育在摇床于30℃培养过夜以230-250转。 </li></ol> 2.成长阶段<ol><li>无菌地将10毫升LB培养基+安培的进入无菌125毫升Erlenmeyer烧瓶，并用100微升过夜W3NN [PDMS]培养到准备LB +安培，使得1接种:100稀释。 <ol><li>对于更大的体积，加入500μl过夜培养物于50ml TB +安培的在250毫升Erlenmeyer烧瓶中，代替LB +安培。 </li><li>另外，使用如步骤2.2第二个"代表"文化。 </li></ol></li><li>在30℃和250rpm孵育在振荡培养箱直到尽头TURE达到中期到晚对数期，由光密度（OD）或吸光度表示:A 600 = 0.8。 1-2小时后，将培养基应该开始出现浑浊肉眼。 <ol><li>无菌传送1 ml培养到一个干净的分光光度计小杯中。 </li><li>使用设置为600纳米波长的紫外可见分光光度计测定光吸收。使用1个毫升其中细胞生长至设定空白（LB或TB）的媒体。或者，使用步骤2.1.3代表文化这一步吧。 </li><li>如果培养尚未达到适当的外径，返回到摇动器，并再次检查每半小时。检查OD更频繁的A 600接近0.8。 </li></ol></li><li>一旦培养达到晚期对数期，由A 600 = 0.8所指出的，在无菌条件下转移培养至无菌50ml离心管中。 </li><li>通过以10,000 xg离心离心培养10分钟收集细胞。检查对于PEllet在管的底部。 </li><li>弃去上清液清除到一个合适的生物危险废物的容器。请勿打扰细胞沉淀。 </li><li>重复步骤2.4-2.5，直到所有的细胞已被收集。 </li><li>重悬在100μlLB +安培沉淀。吸出使用微量或使用涡旋混合器。对于更大的ministring生产，再从暂停50毫升TB +放大器文化沉淀在1ml新鲜LB + amp培养基中。 </li></ol> 3. Ministring生产<ol><li>无菌在125毫升的Erlenmeyer烧瓶中，并热加20毫升LB培养基+安培至42℃。更大ministring生产，制成500毫升之LB +安培的，预热至42℃。 </li><li>转移到悬浮预热网上平台。 </li><li>孵育在42℃和250rpm，直到过去数期，由A 600 = 1.0来表示。在42℃，不要离开20毫升文化过去的1小时。 </li><li>降低温度至37℃，并使培养的阿迪TIONAL 90分钟。为500毫升，离开培养90额外诱导期 - 120分钟，并不会降低的温度。 </li><li>降低温度至30℃并保持培养物在200rpm振荡2小时的额外的温度降档期间。对于500毫升，扩展温度降档至4小时或过夜。 </li></ol> 4.收获Ministrings <ol><li>无菌转移文化进入无菌的50ml离心管中。离心在10,000rpm×g离心10分钟，并检查是否有一个小球。 </li><li>倒出上清液到一个合适的生物危险废物的容器，而不会干扰颗粒。重复，直到所有的细胞已收集。 </li><li>保留质粒提取沉淀与在-80℃下提取后液氮存储中的任何商业开采和内毒素去除试剂盒或闪存冻结。 </li></ol>

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作者宣称他们没有竞争的经济利益。

我们在这里描述为生产使用一步法热诱导协议，它不需要特殊的设备，除了这种处理是在典型的细菌的生长和操纵用于LCC DNA ministrings的一个简单的系统。 DNA ministrings是自由扭转，稳定的线性DNA表达盒，缺乏原核的遗传元件。它们可以代替用于基因转移，报道基因的表达，以及在朝向的转基因表达在真核系统中的效率评估的常规方法使用。 DNA ministrings结合生物相容性和线性DNA载体的优越的安全"迷你"载体提高转染效率的好处。我们的健壮一步法体内生产平台迅速和容易产生的DNA ministrings为任何基因转移应用程序，而不需要多个昂贵的体外处理。 有几个关键步骤确保最佳ministring生产（ 图2和3）。热感应是ministring生产中最关键的一步。完全缺乏ministring生产是最有可能是由于缺乏热诱导（保持在30℃的细胞），其中protelomerase表达的抑制防止了前体的质粒的转化为LCC ministring的。以下的热感应协议，预期大约75％的生产效率。而细胞对数生长期热感应是最佳的。虽然目前在固定相当使用细胞（"杂草丛生"， 图3）ministring PE无统计学差异显著，我们确实观察到几乎整个质粒产量下降了50％。产率将取决于所用的质粒提取协议。对于最佳ministring生产，引发热感应而细胞处于对数期。差ministring生产将最有可能是长时间的温度升档或insufficie的结果nt的温度降档。长时间温度升档气馁，因为这会激活E.大肠杆菌热休克反应15，这可能抑制protelomerase活动，并与质粒稳定性造成干扰。因此，定时，温度降档是高效ministring生产另一关键步骤。如果没有在30℃额外孵化时间，ministring生产效率被认为是非常差（"早拆"， 图3）。这可以归因于的时间为电话表达和活性所需的量。始发噬菌体PY54编码电话protelomerase通常感染耶尔森 ，其最佳生长约28℃，12，这表明30℃也可以是用于电话活动更优化。 该DNA minivector生产系统利用体内平台上生成高质量的细菌无序列的DNA minivectors。修改该协议可能我nclude改变生长培养基，以优化的质粒和蛋白质生产，以促进在生长阶段的细胞生长（TB代替，LB），或增加ministring生产和转换效率。对于较大的培养体积（200 ml和更高），温度降档，并在30℃培养可过夜而对ministring PE严重的负面影响进行扩展。我们已在我们的500毫升文化作为一个例子协议的修改。 DNA ministrings纯化可通过原核生物骨干体外内切酶消化加快。有许多内切酶靶位点（ 例如 ，PI-SceI位，PvuⅠ的，ScaI位）上的前体的质粒，其可被切割以暴露开放线性的原核骨干可用结束时，允许在原核骨架的体外降解。从其它载体物种ministrings分离也可以通过阴离子交换膜层析16来实现的。 与LCC DNA载体生产系统相关联的一个电流限制是用于从其它LCC和CCC产品的DNA ministring的纯化的需要。虽然很容易地用标准的质粒分离方法纯化，所述分离步骤还可以在大规模的设置的缺点。所述ministring分离可以是费时使用AGE如果ministring和LCC原核生物骨干在尺寸太相似了。另外，阴离子交换膜层析可以提供从CCC媒介物种16 ministrings更好的分离。温度升档可以是另一种潜在的限制，因为高温也激活在大肠杆菌中的热休克反应大肠杆菌 ，其负面影响重组蛋白产生17，从而减少质粒率ministring转换。我们在其他地方报告生产平台的优化，以规避和/或减轻热休克18的这种影响。我们目前还优化方法，以消除或减少LCC原核生物骨干污染体内 。 LCC的DNA ministring的染色体整合破坏宿主染色体和细胞科目凋亡。这不仅减少插入诱变诸如肿瘤发生和相邻基因的沉默，从而提高其安全性的潜在的负面影响;致死作用也可以作为敲除中和基因置换的研究没有相关的副作用和一体化的损伤的理想方法。的DNA ministring载体可以向基因用于治疗蛋白质，RNA，抗体，或抗原的体内转移和表达成给定的目标应用或更换造成故障或无功能的等位基因。在简单的一步热诱导体内生产系统允许用于临床或工业应用中容易产生的DNA ministring载体。

的目标是产生线性共价闭合使用简单的和高效率的一步热诱导体内 DNA ministring生产系统（LCC）的DNA minivectors的伸缩量。 DNA ministrings提供一个安全，有效的非病毒性的策略来提供的DNA。他们结合的LCC载体的安全性与DNA小环的效率，并且还提供到病毒衍生的载体更安全的替代不影响转染效率。 为了使转基因递送系统是成功的，该DNA载体必须进入靶宿主细胞和表达编码转基因（多个）。有需要在实践中加以克服，特别是在哺乳动物系统几个细胞的障碍。为了通过网状内皮系统，以避免血清核酸酶和免疫检测退化，DNA载体必须是生物与目标系统免疫兼容。未受保护的DNA很快被血浆核酸酶消化和质粒膜由密集的脂蛋白障碍，使该DNA载体必须能够迅速地横过靶细胞的质膜。一旦在细胞中，载体必须遍历细胞质和穿过核膜以输入的转基因表达的核。非病毒基因传递技术侧重于增强组织特异性和细胞靶向。然而，这些技术的使用常规的质粒的降低转染效率由于免疫原性细菌序列的存在，从而迅速沉默基因表达的1,2-。传统的质粒一般带有抗生素抗性基因在原核系统的维护。然而，这些可能产生的人类宿主的潜在不利影响，或传授给通过基因水平转移的效果自然发生主机菌群阻力。常规的质粒还含有二核苷酸CpG基序2，其可以引发有害免疫反应，潜在地减少或沉默转基因的表达。 因为它们仅包含真核表达盒的，DNA minivectors如DNA小环3，是由于它们的减小的尺寸和缺乏免疫原核元件的，因为它们表现出改进的细胞外和细胞内的生物利用度和改进的基因表达的基因传递一个更好的选择4。减小的矢量大小票价相对于电阻与体内给药到靶位5相关联的剪切力更好。每单位质量的矢量的较高拷贝数的需要较少的转染试剂，从而降低毒性。然而，在随机矢量积分的情况下，进宿主染色体，圆共价闭合（CCC）的载体，包括DNA小环和常规质粒，赋予分子的连续性，因此可能导致插入诱变，其可具有破坏性的后果<s达&gt; 6。比较，线性DNA载体整合破坏染色体并启动的细胞死亡途径，从而去除从增殖细胞群体的突变体细胞，并防止插入诱变7。简约免疫定义的基因表达（蚊）载体8和微型线性矢量9（MILV） 在体外开发LCC DNA载体。蠓载体已在体内表现出高达17倍提高转基因表达相对于传统的质粒DNA载体11，并且已经证明，在疫苗10和癌症8的基因治疗有希望的结果。此外，由于将LCC minivector的自由扭转的结构，较少的转染试剂，需要相比于CCC超螺旋的对应，从而降低毒性7。 我们的增强LCC DNA载体，DNA ministrings，都表现出了超强的表达效率和bioavai7不稳定。此外，DNA的ministrings是在一步法热诱导体内生产系统中，一个权宜和具有成本效益的替代蠓和MILV LCC载体，其需要在体外的多个步骤制备。该DNA ministring生产系统待证明是在体内进行的简单热诱导过程中，使其既具有成本效益和易于扩展。该系统利用了小肠结肠炎耶尔森菌噬菌体PY54衍生电话/ PAL protelomerase重组系统12到最小的真核表达盒从原核表达载体主干分开。我们设计的大肠杆菌细胞（W3NN）的热诱导噬菌体λ启动子，CI [TS] 857 13的控制下，表达电话protelomerase。表达时，电话protelomerase作用于"超级顺序"（SS）位于前体质粒网站中存在PAL靶位点得到LCC产品，从其中DNA ministring然后可纯化（ 图1）。在DNA ministring前体质粒的SS网站还编码为其他重组酶包括Cre重组酶（LOX），TELN protelomerase（telRL）和Flp重组（FRT）靶点，从而有利于从一个前体质粒生产等基因LCC和CCC minivectors的。此外，每个SS网站由SV40增强子（SV40e）序列，其用于改善核易位14在两侧。我们已经在别处证明，连续另外SV40e的逐步赋予的转染效率7相应增加。该前体的质粒（质粒DNA MiniString或PDMS）含有多接头（ 图1），以促进在转录融合的绿色荧光报道（GFP）感兴趣的任何期望的基因的插入。 该DNA minivector技术可能代替用于基因转移，报道基因的表达，并朝向转基因表达的在真核系统中的效率评估的常规方法使用。 DNA ministrings结合生物相容性和线性DNA载体的优越的安全"迷你"载体提高转染效率的好处。我们强大的一步法生产平台，快速方便地生成DNA ministrings任何基因转移的应用程序，并提供了更高的安全性。

<table border="0" cellpadding="0" cellspacing="0" width="1428">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">E. coli W3NN&#160;</td>
			<td style="width:295px;">Mediphage</td>
			<td style="width:268px;"></td>
			<td style="width:492px;">strain for ministring processing: Nafissi &#38; Slavcev, 2012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">pDMS.IRES.GFP</td>
			<td style="width:295px;">Mediphage</td>
			<td style="width:268px;"></td>
			<td>parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">BD&#8482; Difco&#8482; Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher BioReagents&#8482; Terrific Broth</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">BP2468500</td>
			<td style="width:492px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BD&#8482; Bacto&#8482; Dehydrated Agar</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ampicilin</td>
			<td style="width:295px;">MP Biomedicals LCC</td>
			<td style="width:268px;">2190147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ultrapure Milipore Water</td>
			<td style="width:295px;">Fisher Scientific&#160;</td>
			<td style="width:268px;">Z00Q0V0US</td>
			<td>Model: Mili-Q Advantage A10 Water Purification System</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Surgical Gloves</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">(S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Fisherbrand&#8482; Petri Dishes with Clear Lid Raised Ridge (100 x 15 mm)</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">FB0875712&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">30 mL KIMAX&#174; Test Tubes</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89001-448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spectrophotometer Cuvette</td>
			<td style="width:295px;">Sarstedt</td>
			<td style="width:268px;">67.74</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile 15 mL Falcon Tubes</td>
			<td style="width:295px;">&#160;BD Falcon</td>
			<td>62406-200,</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">&#160;Sterile 50 mL Centrifuge Tubes</td>
			<td style="width:295px;">&#160;FroggaBio</td>
			<td>2930-S0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon&#8482; Disposable Polystyrene Serological Pipets 10 mL</td>
			<td style="width:295px;">BD Falcon</td>
			<td>13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon&#8482; Disposable Polystyrene Serological Pipets 25 mL</td>
			<td style="width:295px;">BD Falcon</td>
			<td>13-668-2&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Micropipettor (100-1000 &#956;L)</td>
			<td style="width:295px;">FroggaBio</td>
			<td style="width:268px;">C1000-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Micropipettor (20-200 &#956;L)</td>
			<td style="width:295px;">FroggaBio</td>
			<td style="width:268px;">C200-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Pipette Tips (100-1250 &#956;L)</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89079-472&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Pipette Tips (1-200 &#956;L)</td>
			<td style="width:295px;">VWR</td>
			<td style="width:268px;">89079-460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher Scientific Economy Burner</td>
			<td style="width:295px;">Fisher Scientific</td>
			<td style="width:268px;">03-917Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Incubator Shaker</td>
			<td style="width:295px;">New Brunswick Scientific&#160;</td>
			<td style="width:268px;">M1352-00000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Erlenmeyer Flask (125 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980125 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sterile Erlenmeyer Flask (250 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980250 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">&#160;Sterile Erlenmeyer Flask (500 mL)</td>
			<td style="width:295px;">Pyrex (Sigma Aldrich)</td>
			<td>CLS4980500 Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spectrophotometer</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:268px;">335901</td>
			<td>Model: Genesys 10 Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Floor Centrifuge</td>
			<td style="width:295px;">Beckman Coulter&#160;</td>
			<td style="width:268px;">369001</td>
			<td>Model: Avanti J-E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Benchtop Centrifuge</td>
			<td style="width:295px;">Beckman</td>
			<td style="width:268px;">362114</td>
			<td>Model: GS-6R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fisher Scientific Analog Vortex Mixer&#160;</td>
			<td style="width:295px;">Fisher Scientific&#160;</td>
			<td style="width:268px;">02-215-365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">1 kb DNA Ladder</td>
			<td style="width:295px;">New England BioLabs</td>
			<td style="width:268px;">N3232S</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of double-stranded dna ministrings

我们构建线性共价闭合（LCC）的DNA minivectors经由体内一个简单的平台产生的非病毒基因递送载体的替代，DNA ministrings具有一个升高的安全性，也有效地提供的DNA货物到靶细胞。常规的DNA载体携带不希望的原核序列，包括抗生素抗性基因，CpG基序，和细菌复制起点，这可能导致的宿主免疫应答的刺激。常规DNA载体的生物利用度也受到影响，由于其更大的分子大小。其圆形性质也可赋予染色体整合，导致插入诱变。 细菌序列是从DNA minivectors切除，仅留下感兴趣的（GOI）的和必要的真核表达元件的基因。我们LCC DNA minivectors，或DNA ministrings，是缺乏免疫原性细菌的序列;因此，提高个EIR生物利用度和印度政府表达。在矢量积分的情况下入染色体中，LCC的DNA ministring将致死破坏宿主染色体，从而去除从增殖细胞群的潜在危险的突变体。因此，DNA ministrings提供"小环"DNA的好处，同时消除了不良的载体整合事件的可能性。相比于常规的质粒及其同基因型圆形共价闭合（CCC）的对应的DNA ministrings展示优越的生物利用度，转染效率，以及细胞质动力学 - 因而它们需要较低量为靶细胞的有效转染的阳离子表面活性剂。 我们已经构建了在大肠杆菌中生产的DNA ministrings的是使用简单，快速，可扩展的一步法体内系统诱导的。

DNA提取，残余前体的质粒，LCC原核生物骨干，并且该DNA ministring之后将被恢复。整体的质粒产率和纯度可使用分光光度计进行评估。纯度是使用吸光度的比率在260nm和280nm（A260 / A280），其中的1.9-2.0的比率是最佳的估计。所有LCC和CCC的产品可以通过琼脂糖凝胶电泳（AGE）（ 图2）分开。 AGE形象化ministring生产热诱导后，ministring纯化之前的结果。 ministring生产的定性评估可以在这个阶段比较ministring（2.4 KB）母公司质粒（5.6 KB）（ 图2）带强度进行。 Ministring DNA可以通过以下年龄标准质粒凝胶提取方案回收。 图3总结了可能，同时携带出现在各种条件下ministring生产效率ING出的协议。根据使用制造商的分析程序的DNA带的强度被确定Ministring生产效率。任何类似密度的程序可以被用来分析AGE的结果。 <img alt="图1" src="/files/ftp_upload/53177/53177fig1highres.jpg" width="700" /> 图1:ministring生产概况 （A）的亲本载体含有在转录融合多接头用报道基因，由两个SS站点成帧。骨干编码β-内酰胺酶抗氨苄青霉素，也包含独特的骨干体外消化几截点。主协议的（B）的综述。步骤2）生长阶段和3）Ministring生产的图中被表示，以提供所涉及的步骤的总体示意图，突出协议的简易性。 （C）Ministring感应。热感应失活的温度敏感的阻遏，允许电话表达随后在父质粒SS网站电话活动。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53177/53177fig2highres.jpg" width="350" /> 图2:Ministring产量从协议偏离后 。 1:控制，2:未诱导，3:蔓生，4:Overinduced，5:早期切除。 DNA ministring约为2.4 kb的，LCC骨干为3 KB，而pNN9父质粒是5.6 kb的。 ministring和LCC骨干带的带强度与方案偏离下降，表明较低的ministring产量。从左至右依次为:新英格兰生物实验室1 kb梯度;控制，以下协议;未诱导，没有温度升档;蔓生，温度升档都灵克固定相; Overinduced，扩展温度升档;早拆，没有温度降档。采用欧米茄百特EZNA质粒Mini试剂盒提取质粒。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig2highres.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/53177/53177fig3highres.jpg" width="550" /> 图3:对ministring生产效率（PE）从协议的偏差的影响的结果是3次实验的平均值。 PE是使用HP AlphaImager岁后测量的相对带强度估计。偏差发生在以下的步骤:控制，以下协议;未诱导，在步骤2中没有温度升档;在步骤1.6静止期杂草丛生，温度升档开始; Overinduced，延长在步骤2.3升档温度;早期切除，取出细胞并提取没有在步骤2.3温度降档。 <a href="https://www.jove.com/files/ftp_upload/53177/53177fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Production of Double-stranded DNA Ministrings at JoVE.com

Production of Double-stranded DNA Ministrings

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

双链DNA Ministrings的生产

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

production-of-double-stranded-dna-ministrings

Research

JoVE Journal

Biology

10.3K 次观看.  University of Waterloo. The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings. 

视频: Production of Double-stranded DNA Ministrings

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全基因组阵列比较基因组杂交技术示范

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

科研

生物学

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

慢病毒生产

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA的稳定同位素探测（DNA的SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

迭代优化的DNA双链的SeqA-DNA复合物的结晶

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...