在HCC4017和HBEC30KT细胞系被好心医生约翰·明纳的实验室提供。这项工作是由塞西尔H.和艾达绿色基金李章博士的支持。

注意：当放射性物质的工作，采取适当的预防措施，以避免实验者和周围环境的污染。处理掉所有废物以下本地辐射安全指引。 1.准备细胞<ol><li>种子细胞3.5厘米板，使得所述汇合上测定当天达到80％-90％。 <ol><li>需要注意的是接种汇合为细胞取决于细胞类型和它们的生长速率。当用试剂为一定天数的治疗的细胞，种子细胞，使该汇合是80％-90％的亚铁血红素测量的一天。如果从3.5厘米板获得的细胞的数量不足，则使用6厘米板用放射性的ALA相同量中的下一个步骤。 注意：使用单独的板的建议，因为它更容易处理各个板。 </li></ol></li><li>血红素测量前一天，加0.1-0.3微居里[14 C] 5-ALA和ALA冷达成最后的Concentration的20μM的总的ALA到每个培养皿中的工作区适合与放射性材料的工作，并把板放回孵化器。理想的情况下，孵育用放射性ALA 16小时。 注意：放射性标记的5-ALA需要可能的细胞系之间变化。至少100每分钟（CPM）一种放射性计数，需要尽量减少与计数相关联的错误。最好是确定[14℃] 5-ALA的需要对所研究的细胞系的最低量。对于大多数实验0.1-0.3微居里足以获得良好的测量结果。加入冷的ALA是可选的。 </li></ol> 2.提取血红素<ol><li>放置细胞培养皿在冰上，并将其送往该区域适合放射性工作。 <ol><li>执行整个萃取和蒸发过程中的防爆罩。因为存储甲醚发生爆炸，使用小瓶子乙醚和蒸发任何剩余的液体，不要储存乙醚开口瓶中超过一个月。不要使用丙酮靠近明火和加热器，因为它是高度易燃。商店丙酮的试剂玻璃瓶，因为它溶解组织培养塑料。 </li></ol></li><li>抽吸掉使用移液管的介质。用1ml 1×冷PBS洗涤细胞一次。关闭吸PBS。 </li><li>加入1毫升1×冷PBS到板和收集细胞在用橡皮板的底部。从各板传送所收集的细胞进行标记的预冷2毫升管。收集所有细胞的2ml管。 注：使用新的橡皮每个板各板刮细胞。对于每块板，你会需要四个2ml管，1.5ml试管和闪烁瓶中。 </li><li>自旋以15,000 xg离心1分钟，在4℃。 </li><li>取出PBS中使用吸液管，给短的自旋和除去残留的PBS中。重悬的细胞在100μl1×PBS中。涡旋重悬细胞，并保持在冰上。 </li><li>就拿10＃956;升从步骤5和转移到1.5ml微量离心管中并储存在冰上以测量蛋白质浓度。 注：细胞裂解缓冲液应加入到细胞中，并保持在冰上存货来测量蛋白质浓度。 </li><li>自旋从步骤5其余90微升在5000×g离心30秒，在4℃;完全用200μl枪头取出PBS。 </li><li>加入300微升血红素提取缓冲液（见材料清单），以每管。然后涡旋15秒以混合雪藏了1分钟，重复此3倍。 <ol><li>添加血红素提取缓冲液（HEB）和涡管迅速重悬沉淀，做只有一个或两个管的时间，因为粒料应迅速再悬浮而不会留在HEB太久。一旦HEB被添加到所有的管子和沉淀重悬然后按照15秒的涡流和1分钟在冰上循环如在步骤8.注意粒料可能不会完全进入HEB如果颗粒被留在HEB缓冲器为一再时间，不会很快重悬。 </li></ol></li><li>添加1.2毫升乙醚到每个管，涡旋30秒。自旋以15,000×g离心5分钟，在4℃。 注：乙醚快用完了枪头。为了控制此，吸管乙醚上下一次或两次，这样做有助于保持在吸移管的乙醚。这是一个新的枪头是用来每次重复。 </li><li>用1ml移液器转移顶部（醚）相到新的离心管中。加入0.5毫升2N盐酸，涡旋混合和自旋如步骤9。 注意：这是很难看到醚和HCl相之间的分离。因此，在实验过程中被使用，足以给它一个淡蓝色的颜色在2当量盐酸溶液中添加少量的锥虫蓝的。这将有助于区分这两个阶段。下层相将是蓝色和上部醚相将是无色的。上层无色醚相转移到新的试管，弃去下部的HCl（蓝色）相。 </li><li>重复步骤两名10更多次，以用2当量盐酸洗涤乙醚相。只有顶部醚相转移到新管中每个重复期间，丢弃下层盐酸相。 </li><li>第三次洗涤用HCl后，顶部相用1ml移液器转移到闪烁瓶中。由通过保持样品中的化学罩O / N蒸发乙醚干燥样品。 </li></ol> 3.测量放射性<ol><li>第二天，从步骤2.12加5毫升的闪烁流体向干燥的样品。 </li><li>摇匀混合。通过使用闪烁计数器测量放射性。 </li></ol> 4.计算<ol><li>从步骤2.6用CelLytic M缓冲等量裂解的细胞（见材料列表）和自旋以14000×g离心5分钟，在4℃。 </li><li>取上清液的等分试样，并使用BCA蛋白测定试剂盒测定蛋白质浓度。 蛋白以mg（TP）的合计量=（90微升*浓蛋白在81克/微升）/ 1000 血红素的合成水平（放射性/ TP）=蛋白质皮摩尔/毫克。 </li></ol>

我们什么都没有透露。

血红素起着经由呼吸26在细胞能量的产生的关键作用。改变血红素代谢是已知的各种疾病，包括癌症28,41相关联。血红素合成的抑制已知会导致细胞周期阻滞和凋亡的Hela细胞26,41。它已经表明，高血红素合成水平与肺癌细胞28的进展有关。因此，这将是非常重要的测量在不同条件下的细胞血红素生物合成的水平。这里讨论不量化血红素的总量因此该方法，但它不提?...

血红素，亚铁离子，原卟啉IX的络合物是中央分子用于运输和利用在几乎所有的活生物1-3氧。血红素的独特结构使其能够用作双原子气体和电子的载流子，以及执行各种其它功能1-5。例如，血红素结合氧血红蛋白和肌红蛋白对氧6,7的传输和存储。它也可以作为在细胞色素电子载体呼吸期间和充当用于通过细胞色素P450酶8,9-催化氧化还原反应的电子供体。之一的血红素的最显著特征是，它可以在细胞和分子过程，如基因转录，蛋白合成和微RNA的生物发生4发挥调节作用。例如，它通过控制哺乳动物转录阻遏BACH1的活性和哺乳动物核REC影响许多基因的转录eptor REV-erbα10-15。血红素调节血红素活化蛋白（HAP）1，它起着参与呼吸和控制氧化损伤的基因的活化中起重要作用，响应于血红素或氧气16的激活。血红素也调节基因转录在通 ​​过神经生长因子（NGF）信令3,17-20神经元细胞。它还通过调节血红素调节eIF2α的激酶（HRI）21-24活性调节蛋白质合成在哺乳动物红系细胞。此外，血红素影响关键信号传导蛋白如酪氨酸激酶Jak2的和Src的，这对于适当的细胞功能和细胞生长4,20,25必需的活性。结果发现，在HeLa细胞中的血红素抑制导致细胞周期阻滞与衰老和凋亡26相关的标记物和活化。既血红素不足或血红素水平增加都与人类27严重的健康影响有关。最近的一个分子第二流行病学研究显示高血红素摄入正相关和疾病，如2型糖尿病，冠状动脉心脏疾病和多种癌症，包括肺癌，结肠直肠癌和胰腺癌27,28的风险增加。使用配对正常和癌症肺细胞作者的实验室已经发现，癌细胞已经耗氧量增加，血红素合成和参与血红素摄取和利用氧的蛋白质28的水平。有趣的是，抑制血红素合成减少耗氧量，增殖，迁移和集落形成的癌细胞28。因此，内源性血红素水平波动起着分子和细胞过程3,4,28,29的调节中起重要作用。 在哺乳动物中，血红素的生物合成发生在八个步骤，涉及位于线粒体和胞液4（ 图1）的酶。血红素生物合成论文中线粒体的基质开始与甘氨酸和琥珀酰辅酶A的缩合，以形成5-氨基乙酰丙酸（5-ALA）的通过ALA合成酶（ALAS）4,31催化。这是速率限制步骤中nonerythroid细胞血红素生物合成。 5-ALA然后出口出来到接下来的四个步骤发生，以形成粪卟啉原III（CPgenIII），然后将其导入回线粒体，在那里它被转换成原卟啉IX（PPIX）的胞质溶胶中。最后，铁的一个分子掺入到原卟啉IX（PPIX）以产生血红素，通过铁螯合（FECH）2,4-催化的反应。 血红素生物合成的水平主要取决于ALAS酶被紧紧通过细胞内的铁和血红素4控制的水平。血红素的生物合成可受遗传缺陷，某些矿物质和维生素（ 例如，核黄素，锌），暴露于毒素（ 例如，铝，铅的可用性），某些类固醇（缺氧的，发烧和等级，例如雌激素）32-35。血红素合成的电平被改变的各种疾病状况。血红素下降合成可引起贫血和神经系统疾病3,36。可替换地，增加的血红素生物合成起着某些癌症28,37的发展中起重要作用。血红素已被证明是用于哺乳动物脂肪，红系和神经元细胞4,38-41的生长，分化和存活的关键。例如，血红素缺乏导致通过谷氨酸NMDA（N-甲基-D-天冬氨酸）受体17的抑制轴突损伤在原代小鼠皮质神经元。此外，抑制血红素合成引起程序性细胞死亡的上皮宫颈癌HeLa细胞26,41。因此，测量在不同条件下在各种细胞血红素生物合成水平是研究病因和progressi重要对许多疾病的。 在这里，我们描述了一种快速，灵敏的方法，使用[4- 14 C〕-5- aminolevulic酸来测量细胞内血红素合成的水平。这是一种替代方法，使用55 Fe或59的Fe的其他方法。我们喜欢使用14 C，因为它的辐射非常微弱。相反，需要用铁同位素工作有力的保障。此外，这种方法的目的是测量并以快速的方式在不同的细胞进行比较并行血红素合成。为了测量绝对血红素水平，人们可以使用的涉及使用的HPLC 42,43的先前建立的方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethy ether</td>
			<td>Sigma</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (Hydrochloric acid)</td>
			<td>Fisher</td>
			<td>A481-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Scintillation cocktail&#160;</td>
			<td>MP Biomedicals</td>
			<td>882470</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiolabeled 4-14C aminolevulinic acid</td>
			<td>Perkin-Elmer life sciences</td>
			<td></td>
			<td>Store @&#160; -80 &#176;C</td>
		</tr>
		<tr>
			<td>CelLytic M</td>
			<td>Sigma</td>
			<td>C2978</td>
			<td>Mammalian cell lysis reagent</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Specific reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Component</td>
			<td>Dispense</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Heme extraction buffer- Acetone: HCl:Water (25:1.3:5)</td>
			<td>Acetone</td>
			<td>25ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated HCl</td>
			<td>1.3ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>5ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of heme synthesis levels in mammalian cells

血红素作为辅基为多种已知作为血红素蛋白，例如血红蛋白，肌红蛋白和细胞色素蛋白。它参与各种分子和细胞过程，如基因转录，翻译，细胞分化和细胞增殖。血红素的生物合成水平在不同组织和细胞类型而变化，并改变在疾病状况如贫血，神经病变和癌症。这种技术使用[4- 14 C] 5-氨基乙酰丙酸（[14 C] 5-ALA），早期前体中的血红素生物合成途径来测量哺乳动物细胞血红素合成的级别中的一个。该测定涉及细胞与[14 C] 5-ALA进行萃取血红素结合到血红素的放射性的和测量的孵化。这个过程是准确，快速。该方法测量血红素生物合成的相对水平，而不是总血红素含量。为了证明使用该TECHN的神游血红素生物合成的水平测定几种哺乳动物细胞系。

该方法是用来比较在正常（HBEC30KT）血红素合成的水平与癌（HCC4017）肺细胞。 图2示出了血红素合成中的癌细胞（HCC4017）比正常肺细胞（HBEC30KT）的更高的水平。血红素合成的水平，也测量在正常细胞和癌细胞中的线粒体解偶联剂羰氰-3-氯苯（CCCP）的存在。用10微米的CCCP的血红素合成水平测量之前处理细胞24小时。正如所料，血红素合成的水平（ 图2）中的CCCP的在正常和?...

Watch this Scientific Journal Video about Measurement of Heme Synthesis Levels in Mammalian Cells at JoVE.com

Measurement of Heme Synthesis Levels in Mammalian Cells

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

measurement-of-heme-synthesis-levels-in-mammalian-cells

Research

JoVE Journal

Biology

12.0K 次观看.  University of Texas at Dallas. Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

视频: Measurement of Heme Synthesis Levels in Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

血红素加氧酶1表达上调在刮痧过程中的生物发光成像

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.