这项工作已经由美国国家科学基金会资助的IOB＃0615996。

第1部分：准备线虫的生长介质（NGM）的Petri板实验线虫， 如线虫和 P. pacificus通常在6厘米的Petri板含有线虫的生长介质（NGM）1实验室培养。蠕虫是保持在20 ° C，但可以根据线虫的物种和实验设计，在一定温度范围内（4 ° C - 25 ° C之间）孵育。为了准备与NGM板，标准的无菌技术已被用来防止真菌和细菌的污染。以下是协议准备NGM板： <ol><li>称取蛋白胨2克，琼脂15克，2.4克氯化钠2.72克KH 2 PO 4 2升锥形瓶中，使体积至1 L与水，高压灭菌器，并允许它冷却到60 ° C水浴中。 </li><li>添加0.8毫升的1 M 氯化钙 ，胆固醇（5毫克/毫升乙醇中），1米硫酸镁 。为了防止真菌和细菌的污染，1 ml链霉素（100毫克/毫升）和1毫升制霉菌素（10毫克/毫升），可以被添加到每公升介质。 </li><li>使用Unispense机和保持无菌条件下，倒入所需的板媒体。板应装到约2 / 3 路 。一个标准的直径为60毫米的钢板，需要约10毫升的媒体。不要板块移动，直到琼脂完全gelified，否则会有琼脂表面凹凸不平，使其难以集中的体视显微镜上的蠕虫。 </li><li>一旦干燥，板材可立即接种或储存在4℃，直至与细菌播种彗星。 </li></ol>第2部分：准备OP50细菌和播种NGM的Petri板 P. pacificus是在实验室培养大肠杆菌 OP50应变喂养。 OP50上NGM板的有限增长，从而形成一个薄的草坪，方便的可视化的蠕虫1。要成长的OP50文化，使用LB -肉汤： <ol><li>添加5克，2.5克酵母提取物和2.5 g氯化钠bactotryptone螺丝帽瓶500毫升，用蒸馏水和高压灭菌容至500 ml。 </li><li>同一个OP50单菌落接种肉汤的无菌条件下培养板，让它成长一夜之间在37 ° C。 </li><li> LB - OP50是随时可以使用，可以储存在4 ° C几个月，如果保持无菌。每当种子板块需要无菌一个小烧杯，倒入少量的肉汤，这将有助于防止污染的股票文化。 </li><li>种子板，分配到60毫米直径的NGM的Petri板和漩涡传播的OP50 100-120μL。 </li><li>孵育这些种子草坪生长板在室温下过夜。这些种子的板，现在可以存储长达3个星期在4 ° C。它们存储在一个倒置的位置，有助于防止快速干燥。 </li></ol>第3部分：一挑在本节中，我们介绍了如何挑一个蠕虫。挑用于蠕虫从一个位置转移到另一个。挑30计90％的铂金10％铱丝，虽然一些研究人员可能更喜欢稍有不同的金属成分： <ol><li>剪下约3-4厘米的导线，巴斯德吸管（其尖端被打破到一个较小的长度）的提示，然​​后0.5厘米内入玻璃密封在一个本生灯。从玻璃突起线的长度大约是3-3.5厘米，但可以根据个人喜好而有所不同。 </li><li>拼合线是凸出使用锤子或钳子的结束。然后夷为平地的部分向上弯曲，形成了瓢。 </li><li>用沙纸，以防止破坏性的蠕虫或琼脂，理顺拿起锋利的边缘。 </li></ol>第4部分：采摘蠕虫<ol><li>挑蠕虫，火焰消毒的铂丝和拖细菌草坪扁平尖挑大衣与稠粘细菌的一角，小心不要穿刺或损坏的琼脂表面。 </li><li>使用立体显微镜，很轻轻一刷直到蠕虫被拾起棍子上挑的细菌的蠕虫病毒/蠕虫对粘提示。 </li><li>一旦蠕虫的挑尖，转移它立即到新盘，通过触摸或滑动的挑尖对新盘的细菌草坪和蠕虫抓取。必须小心不损坏板面的琼脂，其他的蠕虫爬进孔，使其难以检索。如果蠕虫停留挑太久，它可能变干。 </li></ol>第5部分：EMS诱变乙基甲烷磺酸盐（EMS）诱导突变在基因组中的2的广谱。突变体可以识别的缺陷，如产蛋，肌肉缺损S，性别决定的dauer形成，行为，和性腺形成。为EMS诱变的协议是类似的 C描述线虫 1。 <ol><li>准备500毫升1N氢氧化钠（或更多）。 </li><li>需4-5，直径6厘米的Petri板的蠕虫病毒，其中包含几百蠕虫幼虫少年第三阶段（J3），洗掉的蠕虫使用2-3毫升无菌每盘M9。 </li><li>在无菌的，一次性的15毫升离心管收集的所有的蠕虫。离心5-7分钟，或在1500 XG直到蠕虫形成一个颗粒。 </li><li>吸出液体，并用2毫升M9的重新暂停蠕虫。 M9的一个试管中2毫升添加20μL的环境管理体系和动摇解散。然后，添加这种蠕虫悬挂和漩涡轻轻2毫升EMS解决方案。 EMS的终浓度为47毫米。注意：EMS是一个非常强烈的诱变剂，并应在通风橱中处理。在这个诱变剂接触的所有材料（手套，滴管，提示），应被视为与1N氢氧化钠不活跃的EMS。双层手套穿在整个EMS诱变过程。根据您的机构的指导方针，处置危险废物的。 </li><li>将离心管中，并用封口膜覆盖的上限;水平放置在通风柜，让孵育3.5小时的蠕虫。管也可以放在低速摇臂。 </li><li>打开管，并用封口膜覆盖的上限;蠕虫下沉的垂直位置，直到孵化的地方。 </li><li>吸出上清液，并丢弃到一个烧杯/烧瓶中含有1N氢氧化钠。这将可灭活该环境管理体系。 </li><li>加入5毫升M9的蠕虫，倒置管的25-30倍，洗5-7分钟，或蠕虫，离心机在1500 XG直到蠕虫形成一个颗粒。吸出上清液，并丢弃到1N氢氧化钠烧杯。 </li><li>重复至少3次洗涤步骤。 </li><li>最后一次洗涤后，重新暂停少量的M9（〜500μL）的蠕虫病毒颗粒，并滴入含有OP50细菌草坪的NGM板出来。在这一点上，你不再需要工作在化学罩。 </li><li>让液体干燥几分钟，然后挑选健康的J4的幼虫转移到新鲜的种子板块。根据您所在机构的危险废物准则丢弃原板。 </li><li>让诱变蠕虫自我受精。使用立体显微镜下，屏幕上的F2代表型为隐性突变体的利益。 </li></ol>第6部分：补骨脂素诱变在本节中，我们描述了长波长紫外线（UV）一起使用TMP的突变过程。 TMP /紫外线诱变的方法被广泛用于诱导缺失突变在基因组 3 。 <ol><li> 4-5，直径6厘米的Petri板收集到15 mL离心管前面介绍的步骤中的蠕虫病毒。 </li><li>添加10毫升M9的反转20次，在1500 XG洗5分钟的蠕虫和离心机。弃上清，重复洗涤，共3次。吸出液体和重新暂停在10倍左右，M9的体积含有30微克/毫升的TMP的蠕虫。吸取20μL到2毫升M9的TMP（3毫克/毫升）。环境管理体系一样，TMP是一种强力致癌物质，必须采取适当的安全措施来处理。为了防止在紫外线照射下正确的眼睛穿的潜在风险，应使用。必须在适当的生物危害袋被丢弃的一次性。 </li><li>在黑暗中孵育15分钟的低转速的摇杆（40芬欧）在室温下管。设置覆盖的管垂直为10分钟，让所有的蠕虫下沉。 </li><li>从巴斯德吸管管的底部取出蠕虫和它们转移到一个非种子选手NGM板。丢弃以下机构的危险化学品的指引上清。 </li><li>保持在暗盘，直到多余的川芎嗪板吸收。 </li><li>使用紫外线强度计，设置之间的长波紫外线源的距离和钢板等，对钢板的强度是500微瓦 / 平方厘米4。删除铝盖，盖，盖，板，含有50秒川芎嗪治疗蠕虫的长波紫外线辐射暴露。 </li><li>盖，在室温下5小时，在黑暗中的蠕虫。 </li><li>挑选和健康的J4的幼虫转移到新鲜的种子板块。丢弃以下机构的危险化学品的指引的原板。 </li><li>使用立体显微镜，屏幕F2代利益的突变表型。 </li></ol>第7部分：代表结果一些在体育pacificus（可以通过EMS或TMP /紫外线）诱变产生的突变表型，如图1所示。每个突变体具有特征性的的表型，野生型动物，很容易分辨。从蠕虫被处理的诱变剂，30 J3/J4从亲代（P0）的幼虫被放置在单独的板块，并让其产卵，这是F1代。从板块表现出健康的F1子代，共1000 F1S被转移到个别板块和F2代表型的筛选。矮胖5,6，不协调6和变压器7表型的动物被发现。 <img src="/files/ftp_upload/2293/2293fig1.jpg" alt="图1" /> 图1。Pristionchus pacificus野生型和突变型的动物。 A. WT雌雄同体B. WT男三DPY突变D。 UNC突变E. TRA突变。前两名面板显示野生型雌雄同体（左）和男（右）的动物，在余下的面板不同的突变体进行比较。

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

突变是一个广泛采用的技术，在各种实验模式生物基因研究。通常用于突变实验，以确定一个基因的2的功能。这里所描述的突变程序可以使用，不仅为体育pacificus，但也为C。线虫和其他相关线虫物种。在这里，我们介绍两种方法，环境管理体系和TMP /紫外线诱变。 EMS是一种化学物质，诱导点突变或整个基因组的3,8小缺失。补骨脂突变（TMP / UV）...

an introduction to worm lab: from culturing worms to mutagenesis

这个协议描述的程序，以保持线虫在实验室，以及如何mutagenize他们使用两种可供选择的的方法：甲基磺酸乙酯（EMS）和4，5“，8 trimethylpsoralen结合紫外线（TMP /紫外线）。线虫是因为其简单的身体计划和交配系统，这是自我施肥雌雄同体和男性可以产生数以百计的每动物后代组成强大的生物遗传学的研究系统。线虫是保持中含有的细菌草坪的琼脂板，可以很容易地从一个板块转移到另一台使用一挑。 EMS是常用的一种烷化剂诱发点突变和小缺失，而TMP /紫外线主要是诱导删除。根据正在使用的线虫的物种，环境管理体系和TMP的含量进行优化。为了隔离隐性突变线虫Pristionchus pacificus，F2代的动物视觉筛选表型。为了说明这些方法，我们诱变蠕虫和不协调（UNC），矮胖（DPY）和变压器（TRA）突变体。

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

与表型的缺陷突变体的筛选是一个简单的方法识别基因功能，在一个给定的生物过程。在这篇文章中我们将介绍如何对文化自由的生活的蠕虫（如，<em&gt; Pristionchus pacificus</em&gt;）在实验室中，并显示两个不同的诱变方法，环境管理体系和TMP /紫外线。

蠕虫病毒实验室介绍：从培养蠕虫突变

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

an-introduction-to-worm-lab-from-culturing-worms-to-mutagenesis

Research

JoVE Journal

Biology

34.2K 次观看.  University of Texas at Arlington. 与表型的缺陷突变体的筛选是一个简单的方法识别基因功能，在一个给定的生物过程。在这篇文章中我们将介绍如何对文化自由的生活的蠕虫（如， Pristionchus pacificus）在实验室中，并显示两个不同的诱变方法，环境管理体系和TMP /紫外线。

视频: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

科研

生物学

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

单卵母细胞的亚硫酸氢钠诱变

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

固体支持基于膜的电

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

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

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

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

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

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

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

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

用小鼠乳腺肿瘤细胞教生物学的核心概念：一个简单的实验模块

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...