Idiotic Business Behavior by Large Banks, A Lesson to Be Learned

When I run my business we run into "situations" or more precisely, difficult customers, when you really want to rather not have that customer instead of doing all the extra work it requires to maintain that customer. But never in my clear mind did we imagine going through something that I just encountered at Bank of America, one of our banks for 20 years. It was a simple situation, they set us up for a type of checking account that somehow stipulated that if we did not maintain $10,000 balance we would be charged a $20 monthly maintenance fee. Since I'd never paid monthly fee for our personal or the business accounts of allelebiotech.com, I probably did not bother to ask why the change. The things got tough in late 2008 to early 2009, I had to scramble all personal funds to not lay off employees, who were going through their own personal difficulties especially the younger ones who were paid by hourly.

Now things are better, I get to pay myself a salary again and pay more attention to my own bank accounts, and one of the things I noticed was that I was being charged a $20 fee at some point during the last 12 months. I asked for a refund for a long term customer's sake, got rejected; then I asked to be removed of any such future fees because my business banks always offer unconditional free checking to all my employees, and got rejected again because "the fee was charged correctly". They did offer a solution, go online open a new checking account since all new accounts opened online will be free of fees.

I did open a new account online, and all its online banking features are exactly the same as the checking account I have been using for 20 years, and online for maybe 10 years. Now I have to redo all my automatic payer and payee accounts, direct deposit, transfer routes... to save $20 a month using exactly the same service. If BOA meant to get a protocol right to charge fees while being able to attract new customers through free checking and encouraging online users to save labor costs, they succeeded, I understand their unspoken business reasons. But it they meant to bother customers to a degree that they would rather leave, they have sure succeeded in a unique way, by which business sense no longer aligns with common sense. The inflexibility they demonstrated through their lower level representatives is quite impressive, I would not allow such handling in my own company. It's simple, the cost to attract and maintain a loyal customer like myself, for 20 years, would be so much higher than somehow partially refunding the $20 fees, or a simple management check to remove future fees without forcing me to open a new account.

I see this as a business real situation lesson, and I know I will be fine doing all my business at my business banks. I am just not quite sure if I should also get rid off all my BOA shares, that may seems like an emotional decisions rather then business though...Oh I also will write to my congressmen and my president not to support more bailout money, they encourage bad business attitude.

傅国涌：林昭正悄悄回到我们的生活中

Note from the blog admin: It is still this Blog's policy to keep repost, retweet to the minimum and maintain this site as a source of original contents. However, this article meets the requirement of being rare in content and unique in style. The topic also suites perfectly the date of today, Christmas Eve 2009.

在鲁迅的故乡绍兴，2009年的中考语文试卷中出现了“林昭”的名字，这是一道成语运用的选择题：

　　下面句子中，加点的成语运用不恰当的一项是( )

　　A.他心高气傲，总喜欢(妄自菲薄)他人，结果可想而知，没有人原意同他打交道，他成了大海里的一叶孤舟。

　　B.从太空回眸我们这颗生存的星球，在(变幻莫测)的白去的飘忽中，它总是散发着一种浅蓝色的绚丽，谁也说不清那究竟是大海还是气晕的颜色。

　　C.林昭曾说：“我们的血是再鲜艳不过，而且是再灿烂不过的墨水，人世间其它一切墨水在这样的墨水面前统统不免(黯然失色) 。”

　　D.凭着健壮的体魄，你可以支撑起一方蔚蓝的天空；凭着旺盛的精力，你可以开垦出一片神奇的土地；凭着巨大的潜力，你可以变得出类拔萃，令人(刮目相看)。

　　林昭，以这样的方式出现在中学生的眼中，也许还是第一次，尽管他们中绝大多数不知道林昭是谁，在匆忙的考试中也顾不上去追问，但，林昭这个名字与他们的生命就这样相遇了，而且是在一次重要的考试当中。我最初是从一个朋友的博客看到这个消息，然后顺藤摸瓜找到了出处，名叫《归忆江天发浩歌》的博客上6月 20日发表的博文《中考卷里的林昭》。

　　这篇博文在我们周围的朋友中引起了小小的惊动，因为林昭的缘故，大家都想认识这个博客的主人。确实，在被杀戮40年后，林昭正悄悄地回到我们的生活中。

　　记得去年冬天，几个朋友一同去乌镇游玩的路上，我偶然在一个朋友的车上听到播放的音乐竟是林昭的《普罗米修斯受难的一日》，这是云南一个摇滚乐队的作品，主唱者刘涛，他们的乐队叫做“腰乐队”，都很年轻，他们把林昭的诗谱曲、演唱，并制成了光盘，在网上流传，我们听了都很感动。通过唱片上留下的电子信箱，我与刘涛联系上了，才知道在西南边陲有这样一群怀抱理想主义情怀的民间音乐人。

　　也是去年，我去湖北一所中学给高一学生做一个《科学家的人文视野》讲座，讲座结束时，我接到许多学生的纸条，有好几个不约而同地问的是林昭、“红楼”的林姑娘，其中一个还将毛泽东和林昭放在一起问：您认为毛泽东是个什么人？对于林昭的人生您有何看法？他们希望多了解一点林昭的历史，这不免让我有些意外，这些90后的学生也开始在探寻真相了。在林昭身后 40年，她的英灵不仅没有消散，而且带着更大的不可战胜的力量回来了，回到我们的中间，与我们这块土地同在。

　　去年5月1日，《南方周末》以《读林昭十四万言书》为题发表我为纪念林昭被杀40周年而写的文章，我曾接到编辑转来一位叫方震的读者来信，他说，读此文“泪如泉涌，泣不成声”，并写下一诗《哀林昭》，请编辑转给我：檄文读罢泪沾襟，漆夜中华一点萤。天下男儿当有愧，林昭青史独留名。

　　林昭已成为民族记忆中一个不可替代的符号，每一个良知未泯的国人一旦了解她的生平、思想和殉难之惨烈，无不对她肃然起敬。80多岁的律师张思之先生将这自己在林昭墓前的留影视为珍贵，90多岁的学者刘绪贻先生惦念着林昭，更多的年轻人把林昭视为20世纪中国精神史上的偶像，“黑暗时代”的一道亮光，中国的基督徒以拥有林昭这样一位殉道的姊妹而深感欣慰。不仅年年的清明节和4月29日的林昭祭日，就是在平日，苏州城外灵岩山上前往扫墓的人也是络绎不绝，生财有道的当地村民甚至形成了为林昭墓带路的小小“产业”，向四面八方前来为林昭扫墓的人收取带路钱，这已经是灵岩山的一道有点煞风景的“风景”。即使去年40周年祭前夕，当局在林昭墓前的树上安装了意为恐吓的摄像头，也没有阻止人们走近林昭的脚步。尽管网上的“林昭小组”、“林昭群”都被解散了，关于林昭的文字也常常被屏蔽。但是，这一切都没有能挡住林昭回到我们生活当中——

　　在山东，一位年轻的女教师海虹，以林昭为原型，写了一部题为《中国红豆词》的长篇小说，2008年6月由中国国际文化出版社出版，本来小说的主角就叫林昭，只是出版时未能通过，无奈地改成了“林萍”，整个故事几乎没有虚构，就是以文学的形式重叙了林昭的生平。

　　在南京，一位年轻的女记者赵锐，为林昭的故事所感动，完成了一本《祭坛上的圣女——林昭传》，已在台北秀威信息公司出版，并于不久前凤凰新华集团在南京举办的“2009年中国南京秋季馆藏图书展销会”
公开陈列展销。

　　在北京，一位年轻的诗人朵朵，写了一出以林昭为主题的独幕剧。也是在北京，以“祭园守园人”自居的朱毅先生，有意倡议为林昭建一个铜像，最好当然是立在北大未名湖畔，退而求其次，则可以放在林昭墓前……

　　戴镣铐的泉，她将永葆青春和甘甜
　　戴镣铐的血，已经自由地流在她体外
　　……受难是她每日的生活，受难是她自愿的选择。
　　……
　　她用盛大的爱来勾兑盛大的苦难
　　在一切黑暗之上，“爱”站立起来
　　“爱”举起的火把永不熄灭
　　……
　　戴镣铐的泉，她将永葆青春和甘甜
　　传薪火的人，她已经来到我们身边

　　诗人三春晖在林昭祭日写下了这首《戴镣铐的泉》，镣铐锁不住泉的涌流，血的涌流，镣铐也锁不住爱的涌流。向死而生的林昭，留下的不是恨的种子，她撒布的是爱和宽恕，那正是一个古老民族所缺少的因子，那是从基督而来的，是通向一个新时代的基石。

　　谷雨过后　大地上的桃花一齐败了
　　落满我的掌心
　　我不是刽子手
　　但我却攥出了一手 淋漓的鲜血
　　……
　　谷雨过后 我从潮湿的江南起身
　　去寻找一支遗失的火炬
　　在石头中　我看见你的名字
　　泼洒着太阳的热情

　　这是诗人涂国文为纪念林昭殉难四十周年写的诗《谷雨过后》，2009年9月，浙江文艺出版社出版他的诗文集《苏小墓前人如织》就收入了此诗，林昭逃过了编辑的“火眼金睛”，这在向来以自律、不出“差错”为金科玉律的浙江出版界，真是一件不容易的事，也许编辑、审稿者压根没有听说过林昭这个名字，所以才侥幸漏网。但是，这样的漏网总是好事，毕竟可以使更多的人听说林昭的名字。

　　在遥远的星空，林昭，正默默地注视着她深深热爱的这片土地。如果能够，她当然不喜欢用血写诗，虽然“人世间其它一切墨水在这样的墨水面前统统不免黯然失色”。她是那么热爱生活，她根本不是政治中人，她不喜欢政治，厌恶那些讲权谋、讲成败的政治，她所争的是是非，是人格，是尊严，这一切在她看来，都是一个人生活下去不可缺少的因素，这一切一旦全部丧失了，那生活当中还剩下什么？她可以为此而死。林昭之死，不只是为她自己争人格，也是为全体国人争人格。今天，她之所以悄无声息地回到我们的生活中，是因为这个时代，我们仍然需要为自己争人格，林昭就是一个最好的本土榜样，她是中国的朋霍费尔，她走过的这条道路已经表明，她已成为古老民族争人格进程中最丰厚的精神资源，因为她提供的不仅有无比锐利的武器，而且有爱的滋润和宽恕敌人的胸怀。幽暗的星空，因着她那双闪烁不定的眼睛，而不再虚空，不再无助，不再绝望。

周末看关于贪官下马新闻随想

最近一年内第三个下马居贪省政协（！）主席的背后故事中出现吴姓高级官员, 感叹政协腐败亦能如此之余，忽然想起大陆出来的人中对于进入官场的热忱。可能一辈子没当过官，也不知当官为何味的普通学术人中亦行此风。认识一位，某次因为有可能有一机会被那位吴姓“有前程”官员接见而兴奋不已，愿千里飞回以“科技”为敲门砖自荐。 可惜该官未成第四代。另一位则以自家人中能有人以学术而进官场（有现成最大先例为榜样）为长远计划，听说某个干部子弟来附近学校上学，即放下长辈、精英架子，主动要为该公子提供关照，尽管黑官是我们作为青年学生时要用毕生理想和一腔鲜血反对的社会腐朽，尽管贪官公子多半儿是在消耗包括自己父母在内的百姓的民脂民膏。不知那个官员到底是谁但好象没有起到什么作用，也许是因为想提供关照者最后也没能关照上，也许该官员没作用力。想起这些并没有低视把当官作为理想追从的人的感觉，微笑之余，忽有一种对生活、思想自由的更多的珍惜，对为人而独立于天地之间无精神束缚的豪爽，和对我在人生选择中有极大启迪的父亲的怀念。

mTFP1 is an excellent FRET donor

Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).

In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.

In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.

Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.

(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.

AlleleBlog Admin, by Nathan Shaner

Video of the month (NEW!): Protein Expression Systems on youtube (http://www.youtube.com/watch?v=n81orbUebsQ) and at our protein expression page.

Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)

New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (http://www.allelebiotech.com/shopcart/index.php?c=197&sc=34), 20 infections, only $249 for a limited introduction time.

ASCB Abstract: Increased rate of reprogramming of induced pluripotent stem cells using high-titer lentiviral vectors encoding multiple cell growth and survival regulatory genes

Objective: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSC) with enforced expression of multiple transcription factors. We aim to improve the reprogramming efficiency using high titer lentiviral vectors encoding additional cell growth and survival regulatory genes.

Methods: Lentiviral vectors encoding multiple cell cycle and apoptosis genes in addition to c-Myc, Klf4, Oct4 and Sox2 were constructed and used to generate iPSC. The iPSC were extensively characterized by immunohistochemical staining and flow cytometry.

Results: Human mesenchymal stem cells can be efficiently transduced and reprogrammed into iPSC using high-titer lentiviral vectors encoding the four known transcription factors. The addition of siRNA suppressing p53 and cell cycle and survival genes including telomerase and BclXL significantly increased the efficiency and rate of iPSC generation. Human iPSC colonies were formed within a week after lentiviral gene transfer.

Conclusions: The protocol for iPSC generation has been improved with high titer lentiviral vectors encoding additional immortalization cellular factors regulating cell cycle progression, senescence and apoptosis. Deletion of the integrated lentiviral genomes using Cre-loxP recombination could increase the safety profile of the reprogrammed iPSC.

more at http://allelebiotech.com/blogs/2009/12/ascb-abstract-increased-rate-of-reprogramming-of-induced-pluripotent-stem-cells-using-high-titer-lentiviral-vectors-encoding-multiple-cell-growth-and-survival-regulatory-genes/

Construction of An Image Library

The American Society for Cell Biology (ASCB) is “pleased to announce the receipt of a U.S. National Institutes of Health Grand Opportunities (GO) grant to build The Cell: An Image Library. The ASCB will be hiring eight cell biologists or microscopists, each at 25% time,” The job description includes, according to an email job posting, “selecting exemplary images and videos and providing metadata for short tags or descriptions as well as longer annotations including technical details crucial for image interpretations. Annotators will select related key words and note biological source, context, item type, etc., in accordance with set guidelines. Annotators will upload images and videos to the Society’s new image library for research and education.” The grant is in the million dollar range.

The need for creating an extensive image library is deservingly recognized by this “GO” grant from the stimulus program awarded to the NIH by the federal government. The difficult part will be to maintain such an image center once the grant runs out. Will it be kept up-to-date and relevant, or left to collect dust on the old images? We wish that the program would be a great success and that the NIH money well spent.

Allele Biotech has applied to the same round of NIH grants with a related proposal that, rather than cell images in general, focuses more on cell differentiation/dedifferentiation through the use of iPS cells. Title: Foundation for “Subcellular Structureome” as Stem Cell Differentiation Parameters. Summary: The key question to be addressed is how to characterize differentiating stem cells along different lineages definitively and continuously, without disrupting or disturbing the differentiating cells. The broad and long-term goals are to find ways of describing stem cell differentiation in more detailed steps, thereby providing methods to predict and direct cell fate commitment.

Aim 1 Create a panel of cells that can be reprogrammed into induced pluripotent stem cells (iPSCs) with fluorescent protein (FP) fusion markers for each organelle

.Human fibroblasts and keratinocytes will be selected from a large collection of primary human cells, based on their ease to grow and transfect, number of potential cell passages, and potentials for reprogramming with induction reagents. A group of 24 subcellular localization polypeptides (LP) and FP fusion protein constructs currently offered by Allele Biotech will be stably transfected into the selected cell.

Aim 2 Characterize the morphological changes of subcellular structures during iPSC differentiation.

Transfected primary cells that stably express subcellular localization marker proteins will be induced with either current retroviral/lentiviral vectors based reprogramming cDNAs, or a non-integrating baculoviral vector under development at Allele Biotech. These cells, 48 lines in total, will be maintained and expanded under stem cell culture conditions, then induced to differentiate into chondracytes or keratinocytes as examples of cell fate. Morphology data will be analyzed and recorded at each known stage and additional “substage” to be defined in the process.

Aim 3 Correlate morphological changes to known molecular properties of each stage and provide a “signature” set of morphological changes for each stage of each lineage

Signature morphological changes, i.e. significantly different shape, location, sub-type, and copies of organelles in a cell compared to its immediate upstream stage, will be correlated to results obtained by standard expression assays at the RNA and protein levels.

Aim 4 Use the morphology parameters to establish more defined stages of cell fate commitments

Data points will be used to create a novel morphology-based cell fate commitment atlas, which will be very helpful in guiding the stem cell and regenerate medicine research at molecular biology, cell biology and physiology levels.

Aim 5 Construct more FP fusions as organelle-specific markers and combine with stage specific gene promoter driven markers

If necessary, we plan to identify more LPs as fusion marker partners after obtaining the initial set of data, and to expand the signature morphology image database. The database can be further complemented with stage-specific gene promoter driven FP images.

Weekly Promotion of Nov 30-Dec 6: 15% off luciferase assay kit ABP-PA-ABLA011 1000 reactions at only $250.00 212.50. Compare it to what you normally pay for firefly luciferase assays and find out how much you are saving.

Reminder: Allele Biotech Spotlight Promo for ASCB Dec 09 Meeting is still on, order by Dec 9th on iPS and FP groups!

New Product of the Week of Nov 30-Dec 6: Allele Biotech’s ProperFold expression vector with fluorescent protein as indicator for proper protein folding, tracking, and purification. pORB-mWasabi+-sIRES-VSVG

Allele Will Receive Its 3rd US Patent On RNAi

On December 1st, 2009 Allele Biotech will be granted its 3rd US patent on using RNA polymerase III (Pol III) for creating RNAi inside mammalian cells. Previously, US patent 7,294,504 was granted to Allele Biotech that covers commercial kits with DNA template components designed for expression of shRNA, miRNA, or siRNA; US patent 7,422,896 further granted claims covering broader designs of using a Pol III promoter such as a U6 promoter for RNAi, including the use of a constitutive or inducible enhancer. The current US patent 7,625,750, protects the use of above technologies in conjunction with arrays, particularly addressable, high density DNA arrays. The RNAi encoding DNA molecules, anchored to the surface through a special peptide, can be transduced via transduction peptide into target cells grown on the array surface. The DNA will then be released from the surface after the completion of DNA transfer by a membrane protease. "This array format for RNAi using the Pol III technology should have higher efficiency and controllability than soft agar embedded siRNAs for transfecting cells, with great potential in large-scale RNAi functional screening and validation. Combined with Allele Biotech’s existing lentiviral vector-based shRNA platform, the addressable RNAi arrays provide us with the best methods available for RNAi screening”, said Dr. Jiwu Wang, CEO of Allele Biotech and the inventor of the patent.



Allele Biotech provides reagent kits and custom services from using its patented technologies in the field of RNAi. The 3 patents issued to Allele Biotech within the past 2 years are so far the only US patents on the methods and compositions of using Pol III promoter for expressing dsRNA-mediated gene silencing. Allele Biotech aims to strengthen its market position by providing superior products and services while actively protecting its intellectual properties. The current strategy includes noting providers and users of existing products that apparently fall under Allele’s patent protection in order to provide reasonable sub-licensing or co-development options.



Allele Biotech is expanding its RNAi capabilities by incorporating the Pol III promoter-driven shRNA cassettes into its popular Phoenix retroviral system and the recently added lentiviral vector system. The RNAi service will also be integrated with Allele’s viral packaging service which offers the best value in terms of per viral particle cost in the market today. High throughput and high content screenings to be conducted at Allele could be further aided by the use of low mutation oligo annealing and wall-less cell array technologies by collaborating with partner companies. In addition, RNAi target design and selection are carried out with an advanced algorism and the most effective empirical rules through Allele’s RNAi services.



Allele Biotech's RNAi technologies were developed with help from the National Institutes of Health (NIH) through several grants. The research team at Allele is currently applying for another NIH project to use these technologies in synthetic lethal screening for cancer therapy. Dr. Wang said that "It is now our goal as well as responsibility to make the RNAi technologies helpful to as many researchers as possible in their pursuit of the best results from gene function studies”.

Fireworks and Music as Czechs Commemorated

Words that will become quotes:

"Many of our citizens who took part in the democratic changes died already with a feeling that they contributed to something that meant a lot," Havel said Tuesday. "In our ordinary, daily lives, we tend to forget our friends of that time -- our comrades, free-thinking individuals."

How many of us can have that feeling?

Allele Biotech Pre-Announces its Product Line in iPSC Creation Using Baculoviral Vectors

As a new approach of establishing novel product lines and generating feedbacks and potential interests early on, Allele Biotech has started disclosing its intended/ongoing research plan on AlleleBlogs or through AlleleNews. This week, Allele Biotech has listed the main points that it intends to accomplish by developing a baculovirus-based iPS cell generating system to complement its existing lentiviral and retroviral vector systems for iPSCs. The plan was part of a grant proposal presented to the NIH during the stimulus grant rounds in mid-2009. There has also been interest on relevant service from Allele Biotech’s customers. The time for market launch is expected to be by the end of this year.

Allele Biotech R&D team welcomes any suggestions and discussions on the research plan and the resulting products. Comments can be made at the blog, through comments on this news, or directly to us through emails.

Take advantage of the brightest GFP for studying gene expression regulations

As most Allele’s customers and website visitors already know, mWasabi is the brightest green fluorescent protein. Unlike commonly used EGFP, mWasabi is a true monomer that does not have the tendency to associate with each other even at high concentrations. It has been validated as an efficient tag in more than 20 fusions located in different cellular compartments.

For studying factors that regulate mammalian gene expression, enzymes such luciferase and lacZ are traditionally used as reporters when operationally linked to promoters and enhancers. Fluorescent proteins are becoming more and more popular for such applications as instruments for reading fluorescence emitted from treated cells are becoming more available. Using fluorescent proteins as reporter eliminates the need for performing enzyme reactions with assay substrate kits. More importantly, fluorescence readings can be taken at any time point on LIVE cells.

Allele Biotech introduces to the market a set of gene expression reporter constructs based on mWasabi and its cyan relative mTFP1, as the new product of the week of 11/09/09 to 11/15/09. Choosing different versions within this vector group, promoters, enhancers, or DNA binding protein binding sites can be easily inserted and their effects of gene expression compared to those of controls.

Promotion of the week: Lentiviral particles expressing commonly used human cytokines at one-time discount.

News article in AlleleNews (to be published Thursday): Using GFP-tag in Immunoprecipitation to study DNA repair pathway factors.

Berlin Wall fell 20 years ago

Read the following writing by Chinese Author 北明:
"１９８９年的最后一天，东、西柏林万人空巷，１９８９年的最后一夜，勃兰登堡门两面，灯火通明，万头躜动。１９８９年的最后一刻，当深色天幕上，礼炮齐鸣，彩花争艳时，人们在欢乐的海洋中奏乐起舞，喜极而泣，仰面沉思、干杯痛饮。两德人民和来自世界各地的游人一齐，在勃兰登堡门迎来了德国历史上的一个新世纪。" Only 6 months earlier young blood was spilling instead of beer and wine. It was the blood of my peers, my neighbors, my friends that awakened some humanity in East German solders and commanders who did not pull the trigger 20 years ago. I feel saddened and proud at the same time. As the saying goes:"Evils prevail when good people do nothing."

Commonly Known Facts About Viral Packaging -That Might Not Be Correct…

Packaging lentiviruses or retroviruses is not a routine procedure that every biology lab performs even if there is need to use it. A viral packaging protocol normally begins with preparation of purified transfer plasmid DNA, a miniprep should be enough for a few transfections. The virus backbone plasmid is either co-transfected into commonly used cells with helper plasmids that provide the essential proteins required for particle packaging, or transfected into established helper cell lines that express the required proteins from integrated transgenes. After incubation of packaging cells for a couple of days, viruses are collected and tittered. Titer determination is somewhat tricky for the inexperienced. Using a control virus expressing a fluorescent protein can make this step convenient.

Commonly known facts:

1) Lentiviruses are packaged at a titer of 10^6 IU/ml without concentrating steps.

This needs update since with more advanced technologies lentiviruses can be packaged routinely at 10^8 IU/ml. With further concentrating, the titer can be easily above 10^11 IU/ml. Retroviruses can be packaged to similar titers as well.

2) Using packaging cell lines gives the highest possible titer

While packaging cell lines (such as Allele’s popular Phoenix Eco and Ampho cells for retrovirus packaging) provides maybe the most convenient method for packaging, the yield will not reach the highest potential. Packaging cell lines may also lose their capability for packaging after continued culturing, requiring periodic selection with antibiotics and functional tests, as we do here at Allele.

3) Retroviruses are always collected in one shot after transfection into packaging cells

If the transfer vector has oriP/EBNA1 episomal maintenance system, such as some of the Phoenix vectors Allele offers, the plasmids may continue to express for up to 30 days. With puromycin selection, the titer of retrovirus produced from Eco or Ampho cells can reach 10^7 IU/ml.

This week’s promotion (102509-103109): 10% off across the board of Allele Biotech’s custom services, for an example, check out our world-leading baculovirus protein expression.

New Product/Service of the Week: Introduction of Custom Viral Packaging Service. Routine titer of 10^8 IU/ml, as high as 10^10 IU/ml, option to include cloning. Signature service ABP-CS-MERV002 provides more than 200 million particles at $7/million particles. These are game-changing prices for the viral packaging service market based on superior technologies!

Biotech Companies Can Now Make Company News Releases Through AlleleNews

AlleleNews publishes the most current, succinct, and to-the-point news and views about biological research that a typical bench scientist would be interested in reading “in-between-experiments”. The full news articles are all original, prepared by senior researchers at Allele Biotech; the short links to top publication news are carefully selected by the AlleleNews staff everyday. The articles are written based on what aspects a graduate student or a postdoc would like to know about a recent paper, instead of what a news agency would typically like them to read. For instance, AlleleNews articles often describe what significance a publication has in which field, how the concept was established and experiments designed, what major distinction it has compared to previous studies or results from other groups, and what areas might be weak points. Less mentioned are the descriptions of the labs, the departments, the institutes and what the officials were quoted as saying about the significance of the discovery. Because of this distinction, the number of readers has been growing at a rapid pace judging from page visits.

AlleleNews site also serves as one of the online platforms for company news releases and user feedbacks. It will be for the benefit of the readers as well as Allele Biotech to further enhance the contents and the functions of this news platform, therefore, a new policy is hereby formally announced: AlleleNews will be open for other companies to make news releases or company announcements. While the AlleleNews administration reserves the rights to make necessary changes, this service will be free to all companies, large or small, with no limits on number of releases. To release any news related to biomedical research or business activities, please email news@allelebiotech.com.
AlleleNews Admin

Q&A About Choosing Modified Oligos

Allele’s New Products of the Week, Oct 19-26, 2009: DNA oligonucleotide synthesis reagents dA, dT, dC, dG controlled pore glass (CPG) beads for oligo synthesis. With previously launched CPG beads and phosphoramidites for modified oligos, this product line now provides the most essential materials for oligo synthesis by university core facilities, company internal oligo production groups, or commercial oligo providers at significantly reduced prices.

Allele’s Weekly Promotion Oct 19-26, 2009: In accordance with the launch of the above new products, all 3’ amino, thiol, Dabcyl, FAM, biotin modified oligos of 50 to 200 nmol scale are offered at unprecedented $10/modification.

Question1:

What do you have available that can be added to the 3’ end of a primer/probe to stop PCR amplification?

There are a few commonly used modifications on the 3′ of an oligo to block polymerase extension, e.g. C3 spacer, amino-modified C6, inverted dT, phosphate. Although no 3′ blocking modifications are 100% effective, the amino-modified C6 offers the best result, leaving1% or less unblocked; phosphate is not as effective of a block, with up to 2% unblocked. We recommend 3’ amino group also because it is less expensive compared to other 3’ modifications if ordered from Allele Biotech.

Question2:

Can you provide 5’ digoxigenin as a standard modification on your oligos?

5’ Dig is typically added by conjugating the digoxigenin group to a 5’ amino added during oligo synthesis. 5’ amino modification can be ordered from almost all oligo suppliers including Allele. You may need to add digoxigenin using a commercial kit by yourself. If you are interested in having Allele Oligo Service perform the chemical linking, email oligo@allelebiotech.com.

Question3:

Is Dabsyl a misspelling of Dabcyl?

DABCYL acid is the abbreviation of 4-(dimethylaminoazo)benzene-4-carboxylic acid. Sometimes DABSYL (4-dimethylaminoazobenzene-4”-sulfonyl chloride) is mistaken for ‘DABCYL’. They do share similar properties as fluorescence quenching agents, with minor difference in maximum absorbance, but can in general be used interchangeably in pair with fluorescent dyes such as FAM. Allele uses Dabcyl as its standard 3’ modification and, by using its own oligo synthesis reagents for adding this group, offers a price less than half of most other oligo manufacturers (check back for pricing updates next week for even lower prices). DABCYL is one of the most popular acceptors for developing FRET-based nucleic acid probes and protease substrates.

Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

New Product of the Week: Anti-GFP Polyclonal Antibody 100ug ABP-PAB-PAGFP10 $175.00.

Allele Biotech has introduced the highly efficient GFP-Trap for GFP fusion protein pull-down, and a monoclonal anti-GFP antibody for detecting GFP-fusion proteins after Immunoprecipitation with GFP-Trap. Just launched this week, the anti-GFP polyclonal antibodies provide an alternative method for analyzing the isolated proteins.

Pre-announcement: Allele Biotech will launch a FAQ and a User Forum online where you can also find common protocols in focus areas and exchange ideas with us or others.

1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×106 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×104/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 105 to 5 x 105 cells, or ~10% confleuce) into plate with feeder cells. Aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium, and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

WSJ article "Deficits and the Chinese Challenge"

It was recommended by fellow members of BayHelix, an association of professionals in the Biotech/Pharma industry most with training in the US after college.

This "interesting article for BayHelix" as mentioned by one member, in my opinion is one of the rather shallow and narrow-minded articles in the WSJ, taking Wall Street Journal Asia as WSJ's Asia edition. World affairs such as wars and survival of nations go way beyond debts and lending. America did not gain power and respect by lending money-- think of the Marshall plan to rescue Germany from the Soviets. America's contributions to both world wars and the prevention of the 3rd one so far certainly included money, lots of it, and blood, lots of that too. Americans took part in so many wars around the world, and the only territories they occupied are those that were used to bury the fallen American soldiers as many of us have already known. Creativity and innovation are results of an open, fair, and competitive society, not a plan of a nation’s leaders or its people’s collective will. Can anyone find another place where competition in a society is more based on one's personal talent, effort, and hard work than in the US? There is no absolute fairness and justice, the American society as a whole is the closest thing to it.

“Fights over health care and climate change are the cultural equivalent of fiddling while Rome burns.” stated in the WSJ Asia article. How about ignoring health care and climate change is historical equivalent of burning Rome by Romes own citizens. When the earth burns, do you really care that much about who still owes you debts?

-----------------
Deficits and the Chinese Challenge
– How United States supplanted the British Empire
By ZACHARY KARABELL
Printed in The Wall Street Journal Asia, October 14, 2009
The dollar's sharp drop over the past few weeks has led to considerable
anxiety about the status of the United States as the dominant force in the
global economy. Closely related to this fear is constant worry about the
rise of China and the evermore complicated relationship between Beijing and
Washington.
Most people are now aware that China is the largest creditor to a heavily
indebted U.S. government. It holds close to a trillion dollars of U.S.
Treasurys and has invested hundreds of billions more in private enterprises
in America. Even though these facts are plainly acknowledged, policy makers
and experts continue to underestimate the full ramifications of this
relationship.
Consider what happened in 1946, when a cash-strapped Great Britain turned to
the U.S. for a loan. For 30 years or more, the British had been consumed by
the threat of a rising Germany. Two wars had been fought, millions of lives
had been lost, and the British treasury was dramatically depleted in the
process. Britain survived, but the costs were substantial.
In spite of its global empire, a powerful military, and an enviable position
at the center of world-wide commerce, in early 1946 the British government
faced a serious risk of defaulting on its financial obligations. So it did
what it had done at various points over the previous decade and turned to
its closest ally for assistance. It asked the U.S. for a loan of $5 billion
at zero-interest repayable over 50 years. As generous as those terms seem
today, such financing had been almost routine in years prior. To the
surprise and shock of the British, Washington refused.
Unable to take no for answer, Britain explained that unless it received
funds the government would be insolvent. The Americans came back with a
series of conditions. They would lend Britain $3.7 billion at 2% interest,
and the British government would have to abide by the 1944 Bretton Woods
plan, which made the dollar rather than the pound sterling the reference
point for global exchange rates and required Britain to make the pound
freely convertible. Even more significantly, Britain had to end its system
of imperial preferences, which meant no more tariffs and duties on goods to
and from colonies such as India. These were not mere financial penalties:
Taken together, they meant the end of the British Empire.
Within two years, Britain had left India and was on its way to decolonizing
throughout Asia and Africa. Unable to compete with the U.S. economically and
no longer able to reap the benefits of colonial trade, Britain's military
shrank and its commerce contracted. It quickly receded from its dominant
global position and entered several decades of economic malaise. In the
1980s, Britain finally emerged as a prosperous country, but it was a shadow
of what it had been in its heyday.
The U.S. replaced Britain as the guardian of the West. As one British
official, Evelyn Shuckburgh, remarked in the late 1940s, "it was impossible
not to be conscious that we were playing second fiddle." And that was
precisely what the U.S. desired. Having supported the British for decades
and become its banker and manufacturer during two wars, at the end of World
War II the U.S. fully intended to supplant the British Empire. The loan
request provided the pretext, but by then the balance had already shifted
and Britain could have done little to reverse the tide.
By 2030—if not sooner—China is likely to surpass the U.S. in the size of
its economy, though it will remain on a per capita basis a much poorer
society for many years after that. Trajectories can change, but the recent
implosion of the American financial system has only accelerated China's
rise.
Given the lesson of the British Empire's demise, it would be foolish to base
current policy on the assumption that China will hit a fatal speed-bump
before it is able to supplant the U.S. And while the level of current
indebtedness is manageable for the U.S.—and in fact tethers the Chinese
closely to the U.S. economy in ways that are arguably beneficial for both
countries—the fact that these economies are currently bound together does
not mean that their interests will always be in sync.
Here, too, the British analogy is sobering. For decades, the relationship
between Britain and the U.S. was mutually beneficial, though the Americans
resented being treated as junior partners. As tension festered, the British
were consumed with the more immediate threat of Germany. But in the end it
was the U.S. that delivered the knockout blow.
The Americans have not had to deal with a true economic rival since the
British more than half a century ago. America today is as unaccustomed to
global economic competition as the British were at their apex. The U.S.
often seems lumbering and ill-suited to the demands of economic rivalry.
The only way to avoid Britain's fate and meet the challenge of China is to
reinvigorate economic life. This is a multiyear endeavor that must be done
primarily through innovation, not legislation. America needs to retool its
domestic economy to build on the global success of many U.S. companies. It
must focus on inventing new products and generating new ideas, rather than
defending the rusty industries of yesterday. Fights over health care and
climate change are the cultural equivalent of fiddling while Rome burns.
China thrives because it is hungry, dynamic, scared of failure and convinced
that it should be a leading force in the world. That is why America thrived
a century ago. Today, such hunger and dynamism seem less evident in American
life than petulance that the world is not cooperating.
The U.S. is in danger of assuming that because it has been a dominant nation
on the world stage, it must continue to be so. That is a recipe for becoming
Britain.

Intracellularly Expressed Camelid Single-Domain Antibody (VHH) Counteracts Cytotoxicity Seen in Agricultural Epidemics

The advantages of camelid antibodies with only the heavy chains have been exploited for therapeutic use or as novel research tools such as immunoprecipitation trap (e.g. GFP-Trap). These so-called nanobodies are small in size (14-15kD), highly stable, and capable of binding to epitopes that traditional antibodies are normally not able to bind. Doyle et al. published a paper in JBC last week demonstrating that when expressed inside cells (intrabody), the VHH fragment can provide antitoxicity protection against deoxynivalenol (DON) or 15-acetyl0deoxynivalenal (15-AcDON), common toxins involved in agriculture infection Fusarium.



Camelid antibodies against low molecular weight 15-AcDON were isolated by immunizing llama with 15-AcDON-BSA protein conjugate as published earlier by the same group of researchers in Canada. As demonstrated previously, the small nanobody derived from camelid antibody can be highly expressed inside target cells without causing much cytotoxicity by itself. Images generated by confocal immuno-microscopy showed that VHH is evenly distributed throughout the cytosol. The antitoxin effects were specific, effective, and apparently dose-dependent.



This report, albeit using a yeast model system instead of natural targets of the relevant plant disease, opens doors to increased tests of using VHH fragments for broader applications in agriculture and more fields other than therapeutics.


Doyle et al. 09-2009

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.045047

Nobel Prize in Medicine Awarded to Discovery of Telomere and Telomerase

Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak are credited with discovering how telomeres work and the function of telomerase. “As cells divide, chromosomes need to be replicated perfectly. Work by the researchers determined that telomeres protect DNA from degradation in the process, and that telomerase maintains the telomeres,” as reported by CNN.

Carol Greider was a student of Blackburn, and Szostak collaborated with the Blackburn group 20 years ago and has since left that field. Still remember going to Blackburn’s seminar as part of the molecular biology seminar series at USC in the early 90’s, and reading Szostak’s papers on aptamer selection while designing RNA aptamer selection schemes (SELEX) to find substrates of pre-mRNA splicing factors.


Product related note: Human telomerase gene TERT is provided on lentiviral vectors to increase efficiency of generating iPS cells.

Allele Introduces New Version of BVES with Insect Specific IRES

Internal ribosome entry site (IRES) can be used to initiate translation of a second open reading frame (ORF) of an mRNA, providing the benefits of: 1) avoiding promoter competition in a dual promoter situation; 2) having controlled ratio of expression of two proteins; 3) placing a dominant selection pressure on the entire bicistronic mRNA and hence the maintenance of the transgene when a selection marker is placed as the second ORF.

IRES elements are located mainly in RNA viruses except certain mammalian and insect mRNA molecules. Only one DNA virus has so far been found to contain an IRES, the while spot syndrome virus (WSSV) of marine shrimp. This IRES, compared to a very few other choices known to function in insect cells such as the IRES from Rhopalosiphum padi virus (RhPV), has strong translation initiation activity (~98-99% in reference to cap-dependent initiation), insect cell specificity, and encompasses only 180 base pairs.

Allele Biotech, with its acquisition of Orbigen, is a major provider of BVES products and services with more than 10 years of experience. Allele’s featured New Products of the Week* this week are WSSV IRES containing baculovirus vectors, the sIRES (for Strong IRES from Shrimp virus) series plasmids. Currently one version is pOrb-MCS-sIRES-VSVG for pseudotyping baculoviruses (within the Emerald Baculovirus for Mammalian Expression series), with pOrb-mWasabi-sIRES-VSVG as a fluorescent protein control; the other is pOrb-MCS-sIRES-MCS for cloning a custom second cDNA. New versions in the future will include IRES driven mWasabi and other commonly used selection markers.

With a current research project for the National Cancer Institute (NCI) within the National Institutes of Health (NIH) involving development of modified BVES and mammalian protein expression and purification systems, Allele Biotech expects this product line to continue its expansion at a fast pace.

* Allele Biotech announces at least one new product every Wednesday through news release at AlleleNews or Allele Blog and social networks.

Allele Biotech Receives Funding from the National Cancer Institute (NCI) to Produce Cancer Antigens

Cancer formation is a heterogeneous and complex process, involving many factors and cellular signaling pathways. There are more than 1,200 potential cancer biomarkers identified in the literature by a 2006 review, which, if analyzed in multiplexicity, may provide the best potential for reliable and early detection of cancer. Many proteins including most cancer antigens become post-translationally modified (PTM) during the “secretory process”, which involves of a journey from their site of synthesis in the rough endoplasmic reticulum (ER), through the Golgi apparatus and then to various cellular and extracellular destinations.

Examples of protein modifications include glycosylation, phosphorylation, acetylation, and amidation. Of these, the most complex procedure is glycosylation, involving several enzymes. There are increasing demands for these glycosylated human proteins in good quantity, purity and affordability by the scientific community to perform fundamental and clinical studies in relation to cancer. Such proteins can not be expressed in bacteria or yeast because those cells do not carry out equivalent PTM as in mammalian cells. Allele Biotech has chosen a modified baculovirus expression systems (MBVES) as the main method for producing glycoproteins and the proposal was chosen by the NCI for funding of $150,000, approximately 75% of the cost of producing 10 glycosylated cancer antigen proteins in the first phase of 6 months. The remaining funding of ~$50,000 mostly in indirect costs will be covered by Allele’s own funds. This SBIR contract will be partially subcontracted to the University of California , San Diego (UCSD) during the glycan analysis phase. The work will be performed in Allele’s San Diego facility and UCSD’s GlycoTechnology Core facility.


This news is released by Allele Biotechnology & Pharmaceuticals, Inc. through AlleleNews, for in-between experiments

The economy recession is most likely over, says who?

The economy recession is most likely over, or so says the federal reserve chairman Ben Bernanke. Do you feel it? Are you seeing increased job opportunities when you leave your current lab or security if you have a post-postdoc position? In our industry, where the health of the economy is mostly measured by research budgets of individual labs or research groups, occasionally by budgets for contracting or licensing fees, the change, if any, is still hard-to-find. But hiring at academic institutes like UCSD seems to have picked up lately, probably due to addition grants from the Obama administration’s stimulus programs. At the same time, individual NIH R1 grants have been creeping up to easily around 1 million a year, program grants 3-5 millions. With more stimulus money kicking in to academic labs this fall, it is expected that the situation will further improve. Comments welcome.

Notes about recent jobs in Pharma/Biotech: since our last blog about massive Pfizer layoff of scientists in 02-09-09, a major layoff in the big pharma sector came from Merck, which announced on 06-11-09 that it would cut 16,000 jobs after completing its merger with Shering Plough. On 09-14-09, Eli Lilly reported job cots of 5,500 or roughly 14% of its work force. There are areas in the country where people report about the economy as “I went to 2 grocery stores and 3 discount department stores over one weekend, and you could do cannon shooting practice in there without hitting a person.” Again comments welcome here, if you believe in a turnaround, or it is all doom and gloom to you. Btw, the History channel has been cranking up the 2012 theories for a couple of months now, if you like the doom and gloom theories.

Note added in proof: As reported in Science yesterday, “a new analysis of the grantsmaking process at the National Institutes of Health (NIH) lifts the veil on how many grant proposals are funded even though they fall below a cutoff based on peer-review scores…at least 19% of NIH’s basic research portfolio is funded for reasons that go beyond quality.”

Retroviral Vectors with Integrated oriP/EBNA1 for IPSC

The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.

By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.

The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.

Difference between hESCs and NSC-Derived Integration-Free iPSCs

By introducing Oct4 and Nanog into human fetal neural progenitor cells, the Muotri lab at UCSD, in collaboration with colleagues from the Yeo and Gage labs of UCSD and Salk, was able to create induced pluripotent stem cells (iPS cells) using the oriP/EBNA1 episomal system to introduce the aforementioned Oct4 and Nanog. While it has been shown by others that with less differentiated cells, such as progenitor cells, it is relatively easy to reprogram using two, or sometimes even one, of the commonly used iPSC generating factors (Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin28) as reported in the AlleleNews article “iPSC generated by using a single reprogramming factor” on August 3rd, 2009, the new publication in PLoS ONE analyzed in detail the differences between embryonic stem cells (ES cells) and iPS cells.



There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:

(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.



Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076



Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.

Effective Concentrations and Effectiveness of siRNA

RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.

RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.

HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.

Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.

Publication by Allele Scientists: A Tale of Twin Functions

A paper published by PLoS One on 08-15-09 demonstrated one RNA binding factor can bind to two completely different target RNA sequences while play leading roles in general splicing machinery and backup roles in alternative pre-mRNA splicing.



In Drosophila, sans-fille (snf) encodes a ~25-kDa protein that was found to carry the functions of two human proteins U1A and U2B”, components of general splicing machinery U1 and U2 snRNPs (small nuclear ribonucleoproteins), respectively. The SNF protein is able to do the double duties by binding to U1 RNA, the RNA component of the U1snRNP complex alone, and the U2 RNA of U2 snRNP if helped by another U2 snRNP -specific protein.



Busy as it sounds, snf has also been known for a long time to be a major participant of the fly sex determination pathway. When the master sex determination gene Sex-lethal (Sxl) is weakened by mutation or in the germ line before SXL fully takes over sex-specific development, snf is required to maintain appropriate splicing patterns of sex-specific transcripts such as Sxl.



How does a general splicing machinery component work specifically in regulated tissue-specific alternative splicing events?



The PLoSOne paper by researchers from Allele Biotech in San Diego and Peking University in China showed that the SNF protein can directly bind to elements of the Sxl pre-mRNA, in a way different from its binging to U1 RNA. Like many RRM (RNA Recognition Motif)- or RBD (RNA Binding Domain)-containing RNA binding proteins, SNF has more than one such conserved RNA binding motifs. Hu et al. in Bin Xia’s lab showed that SNF uses just one RRM to bind to U1 snRNA, but both RRMs to bind to consecutive U-bases on the Sxl pre-mRNA. The result was obtained through NMR analysis of SNF bound to RNAs. This structure-based conclusion agrees well with molecular biology results that showed a longer than usual RNA segment, e.g. ~16 U-bases, is needed for SNF to bind by Jiwu Wang, a co-senior author of the publication.



The long U-runs can be separated into two 8 U-base segments, each perhaps recognized by one RRM. More interestingly, from a mechanical point of view, the two segments can be separated by at least 120-nucleotide distance and still function as effective SNF binding substrate. Functionally, given that many ~8 U-runs surround the alternative splice site on the Sxl pre-mRNA, which are shown by Jiwu Wang’s earlier publications, to be cooperative binding site for the master sex determination factor SXL, it is intriguing to model that SNF binds to these same RNA elements for the same functions. But instead of binding to 2 U-runs cooperatively (therefore much more strongly) as SXL does, it needs 2 runs to just bind to the alternative splice sites. But when SNF does bind, the two U-runs may be used to loop out an entire region of RNA (such as an exon) by one SNF protein, providing effective regulatory functions. This could give a nearly perfect explanation as how SNF can help sex determination only when needed.

For more similar news: http://www.allelebiotech.com/News, or blogs http://allelebiotech.com/blogs/

Hu et al.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006890#aff4

iPS Cells: Feeder Cells

Allele’s entire iPSC product line is designed for the ease of the researcher. Each component in our iPSC catalog will shave priceless time off your protocol by eliminating the tedious steps in iPS induction so you can get down to work.

Allele is adding a major component to its iPSC line: pre-irradiated, ready-to-use, system specific, bFGF-Producing Feeder Cells for iPSC propagation!

Using Allele’s bFGF-Producing Feeder Cells avoids the usual problems associated with MEF cell lines. They are maintained at low passages, come pre-irradiated and ectopically express bFGF so there is no need to supplement your medium with additional growth factors.

Additionally, Allele Biotech is introducing human fibroblasts to the market for iPSC work. MEF is good for mouse iPSC reprogramming but human fibroblast feeders are preferred when creating human iPSCs due to their secreted factors. Propagate human iPSC with greater efficiency while eliminating non-human cells for therapeutic use of human iPSCs!

As always we encourage customer feed back. We are interested to hear about your stem cell work, needs, and requests for new products. We also welcome those who have new ideas and potential products to collaborate with us. We are here to help advance your research and get your technologies to the public.

If you are enjoying AlleleNews and AlleleBlogs: come back and check out our new Forum and FAQ Sections soon to be added to our blogs for quick product/service related exchange and messages of more user control.

Allele Annouces New Products Based on Camelid Antibodies

090209 San Diego—Allele Biotech formally announced today that it has signed an agreement to distribute products from ChromoTek GmbH, a German company with a focus on camelid antibody fragment based precipitation and detection reagents. Single-domain antigen binding fragment, also called VHH or nanobody, can be derived from heavy chain-only antibodies produced by animals in the camel family. The small size and special structures of VHH enable their efficient binding into areas not normally accessible to larger IgG antibodies. Although GFP is a commonly used tag in fusion proteins for imaging, it has not yet become a widely used tag for precipitation. With the introduction of the first VHH-based research reagent GFP-Trap, GFP-fusions will become a desirable tool for pulldown. GFP-Trap is immobilized anti-GFP nanobody, which with a simple procedure, can result in quantitative depletion or isolation of GFP-fusions.



Applications of GFP-Trap may include ChIP-CHIP, CLIP, co-IP, enzyme activity analysis (see Allele Biotech’s product group main page for sample publications). Other products such as anti-RFP and anti-GFP monoclonal antibodies that may be used after GFP-fusion precipitation are now also available from Allele Biotech. “VHH fragments have great potentials in both therapeutic and basic research,” said Allele’s CEO Dr. Jiwu Wang, “The agreement will significantly strengthen Allele Biotech's position in the antibody field”. Allele Biotech started with a grant from the NIH in 2000 to develop ways to display and select antibodies. It participated in a collaborative project on yeast display for selecting antibodies against cancer antigens in 2007 for the NCI. After acquiring Orbigen in 2008, Allele has thousands of antibodies in its product line.

人是可以被驯养的吗？

这里通常不转贴，但看到一段张轲风08年的文章引起了对电影的一点一点想法。影响过90年代成长过程的电影不多，张艺谋“活着”和其它几部可算在其中。其人性的表达和对最底层人民的理解是前所未有的。且表现手法不夸张，不拖带不必要的“艺术”累赘。快进十年，按张轲风的说法：

“在专社会里，如果没有英雄，没有‘核心’，人们会感到恐慌，会故意呼吁出一个“核心”，袁世凯的上台就是证明。从这个意义上说，张艺谋定是一个斯德哥尔摩综合症患者，他通过《英雄》这部影片表达了对秦始皇这个国家“绑匪”的倾慕，并且主观地将当时的六国人全部假想为呼吁统一的斯德哥尔摩综合症患者。其实，他搞错了，七国时代的人还不是斯德哥尔摩综合症患者，心中没有呼唤“核心”来绑架天下的冲动。秦始皇对天下人的劫持是中国人患斯德哥尔摩综合症的开端。”

在什么样的荣华和舒适面前，人如张艺谋者会将自己扭转得如此完整和彻底？

原创Allele Biotech Outpost, more blogs at www.allelebiotech.com/blogs

Immunoprecipitation Tags

Immunoprecipitation is a process of isolating a protein as an antigen by using antibodies against it. It is a powerful tool for studying proteins in biological samples and, in case of Co-IP (meaning immunoprecipitation of complexes containing a known antigen), for analyzing protein-protein interactions. Similar technologies such as chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), or crosslinked and iImmunoprecipitation of RNA-protein complexes (CLIP) aid analysis of protein-DNA or protein-RNA interactions.

The major obstacle for achieving effective immunoprecipitation is the difficulty of finding usable antibodies against a target of interest. A common practice is to use tags that are fused to the C- or N-terminus of the target protein, thereby any validated, commercially available antibody can be used for co-IP in different experimental systems. However, caution must be exercised against potential interference of biological functions from the added tags. In general, one should choose tags that have been tested in many situations and proven non-interfering; still, each biological system is different. Independent validation or supporting data should be used when interpreting results from tag-based co-IP.

Tags are often selected based on high quality and commercially available antibodies. Most commonly used tags include: FLAG, Myc, HA, V5, T7, and His, which are quite small in size and in theory less likely to interfere. GST and GFP are in between 20-30kDa, but they are well documented to form self-contained and stable structures independent of their fusion partners and proved to not interfere in many cases. GST can bind to glutathione beads directly, therefore a top choice for pulldown experiments. GFP or other FPs as tags have the advantages of being also a visualization module to follow the protein both inside cells and during pulldown. However, previously available anti-GFP antibodies, either polyclonal or monoclonal, are not comparable to those against other tags, thereby limiting the use of GFP as fusion tag in pulldown experiments.

GFP-Trap, a recent addition to anti-tag antibodies, is an E. coli expressed, single domain fragment derived from camelid heavy chain antibodies with much higher stability, specificity, and affinity, making GFP based pulldown quantitative. This recent advancement should make GFP in line to become the most suitable tags for many aforementioned precipitation experiments.

A personal mission statement on 43rd birthday

I had a great bd with people most important to me: my family and my best friends' at Disney, during also a 20th anniversary college class reunion. Except my parents and siblings, these are the people who know me best.

Thought I should put some summary on how life's been so far and where it may go next. As a person running an operation, I'd like to put it in a mission statement format, a personal one.

Do research, to satisfy personal curiosity about science in my fields, and contribute to the knowledge pool to help mankind and the environment; do business, support those surrounding me, and support more that surround them; voice against tyranny, help those who can't help themselves and those who feel hopeless in this world.

Jiwu, CEO of Allele

Francis Collins On the Job

Dr. Collins did a town hall meeting style announcement his first day as NIH Director on Aug 17th, 2009. He laid out his view for the NIH: more funding (good), encouraging young scientists (good, average age for first own funding for US biologist is 42, not good), and staying open in communication with society it is serving.

The NIH has $30.9 billion budget for 09 and 2010 thanks to the stimulus addition of $10 billion/year. However, it will feel dried up after two years if the budget plan remains as is. The Obama administration does not seem to want increase the basic research but instead focus more on health care management.

Collins is a well admired director and established scientist. However, it may be a little concerning that he might be too much into “big science” and organized efforts. I don’t know what they teach in graduate classes now but from what I was told 20 years ago curiosity-driven science is the best science and that was what got the US to the dominant leadership in biomedical fields.

Talking about nurturing young scientists, big programs and big labs controlling most grants by proposing big science seem trendy these days. The fight to become one of the big guys in a small, crowded field is a really daunting path for young researchers to tread. The big guys have the say from publication to funding and often times the unpleasant thought and bitter taste of competing against a scientific juggernaut turn young researchers away.

more at www.allelebiotech.com

Camelid Antibodies--background

When it was discovered that animals in the camel family produce antibodies with no light chains, the idea that a single-domain fragment can bind as well as a full 4-chain antibody formed a breakthrough. So far it has been a relatively less known one.

Smaller antibody fragments have been tested for therapeutic uses because classical IgG antibodies are too bulky to penetrate tissues well, and very expensive to produce. Different combinations of antigen-binding variable regions are used, e.g. scFv, Fab, diabody, all to some degree of success. In comparison, the N-terminal domain of camelid antibodies, termed VHH domain, represents a naturally evolved, only 13-15 kD in size, fully functional target binding fragment with many advantages.

The only other known species outside camelidae family that has heavy chain antibodies is particular cartilaginous fish, nurse shark. Although the arrangement of CDRs is somewhat different between the camel and shark heavy chain variable regions, they share many characteristics such as extremely high stability (maintaining functions after100 C heat and extreme pH treatment).

Accumulating reports have demonstrated the therapeutic potentials of camelid antibody-based fragments in treating cancer, neural diseases, even use in hair dandruff preventing shampoo. For basic research, the tiny antigen binders can be used as tools for quantitative pull down with unmatched efficiency, recognizing previously inaccessible enzyme cleft as antigens, and providing libraries for binding partner selection.

Allele Biotech has been working on display antibody selection from its early days through an NIH grant, and recently carried out an NIH/NCI contract for scFv yeast display in collaboration with AvantGen. By working with Chromotek on camelid antibody products, we hope to combine our superior fluorescent proteins with the best antibody candidates and display technologies to move the capture and signaling fields forward in significant ways.

The product line will be formally announced by AlleleNews shortly.

Allele’s Online Community

Allele Biotech’s participation in social networking sites (Twitter, Facebook, and MySpace has been successful over the past few months! We initiated contact with our customers via these websites not only to provide an easy way to let people know about our newest products and promotions but, most importantly, to ensure better customer service through an easily accessible forum for questions, comments, and yes, even criticisms. We are not like those other companies that are so large that your business and opinions do not matter to us. We at Allele Biotech need and appreciate our customers and reward your patronage and participation in these social networking forums with special promotions and customer service that really lets you know we value your online community membership. Your research goals are our research goals!

Several times a week our tweets will inform you about great deals like FREE SHIPPING on select products ordered within a specific time frame.

Our regular blogs found on all three of our sites are used to converse on a variety of topics from SBIR grants to fluorescent proteins to skin care!

On facebook and myspace you can submit technical questions on protocol or products and receive SAME DAY answers; you may also send comments and suggestions for improvement which will be seen by our head scientist and executives. Your opinion counts at Allele! We began this networking concept as not only a way to better reach our customers but, more importantly, as a way for them to better reach us.

Become a friend, fan, or follower to any one of the sites today and receive a $30 discount off your next order!

Why Allele?

Allele provides you with tools that you will find very helpful. The two main motives for Allele developed products are:
1) To incorporate the most advanced technologies in the field
2) To provide equal utility as other companies’ equivalent products at a much more reasonable cost.

How did we do it? By developing technologies internally, in most cases with government grant funding, by in-licensing others’ discoveries, and by listening to you, our customers.

What else do we do? Conduct basic curiosity-driven research just like most of our customers. It helps to stay on the edge and connect to the community.

Use of fetal bovine serum (FBS) Part I, choosing your batch

The maintenance of cell, tissue, and organ from various sources in ex vivo cultures is one of the most important aspects in the bioscience, medicine, and biotechnology industries. Successful cell cultures are largely dependent on the use of fetal bovine serum (FBS) as a common supplement. FBS is an extremely complex mixture of unknown composition. It is found to provide:
1. Growth stimulating and regulating factors for cell growth, proliferation, and differentiation
2. Various protein components for cell attachment and spreading
3. Transport factors for carrying other molecules
4. BSA, antitrypsin, apolipoproteins, antitoxin factors, and other stabilizing factors
5. Vitamins, fatty acids, lipids, and others molecules that can have eff¬ects on pH, free radical scavenging [1], cell signaling, etc.

When choosing FBS, one needs to be keenly aware that the serum is a supplement of still unknown composition, which is known to have the following problems:
1. It contains ambiguous factors exerting eff¬ects on cells that are difficult to analyze [2].
2. The batch-to-batch variability complicates culture conditions and data interpretation [3].
3. It is an animal product that may contain endotoxins, hemoglobin, and factor adverse to intended use of culture.
4. It may carry contaminants such as microplasmas, fungi, bacteria, viruses, and prions.
5. The high protein contents of FBS make downstream processing of pharmaceutical production difficult and costly [4].

It is therefore important to find and test a supplier or even batches for one that works well with a particular cell type, especially when one has just started working with the cells. AlleleVaid FBS is an excellent choice for most of your cell culture needs.

References
1. Kume, T., N. Asai, H. Nishikawa, N. Mano, T. Terauchi, R. Taguchi, H. Shirakawa, F. Osakada, H. Mori, N. Asakawa, M. Yonaga, Y. Nishizawa, H. Sugimoto, S. Shimohama, H. Katsuki, S. Kaneko, and A. Akaike, Isolation of a diterpenoid substance with potent neuroprotective activity from fetal calf serum. Proc Natl Acad Sci U S A, 2002. 99(5): p. 3288-93.
2. Barnes, D., W.L. McKeehan, and G.H. Sato, Cellular endocrinology: integrated physiology in vitro. In Vitro Cell Dev Biol, 1987. 23(10): p. 659-62.
3. Ye, X. and R. Lotan, Potential misinterpretation of data on diff¬erential gene expression in normal and malignant cells in vitro. Brief Funct Genomic Proteomic, 2008. 7(4): p. 322-6.
4. Wang, Y., G. Lu, W.P. Wong, J.F. Vliegenthart, G.J. Gerwig, K.S. Lam, G.J. Cooper, and A. Xu, Proteomic and functional characterization of endogenous adiponectin purified from fetal bovine serum. Proteomics, 2004. 4(12): p. 3933-42.


Part II, work to replace FBS altogether, check back to see the post here later.

AlleleValid FBS (www.allelebiotech.com/allele3/TC_FBS.php) batches come from North American sources that Allele Biotech has access to inspect, and each batch is tested on different insect and mammalian cells in actual experiments scientists perform daily at our own facility.

Our Message to Our Followers

What we at Allele Biotech see ourselves doing in the sense of fulfilling an obligation to the society and our peers in scientific research: Salvation and success through innovation and diligence, reaching for a place of efficient sustainability where monopolies do not win and ordinary people have the chance to realize their dreams.

We don’t believe in dominance by a few big boys, because we don’t believe that they can provide researchers with the best value. We want people that deal with us to see that there is room for development by an individual or a small group of highly dedicated and talented persons, as your own group in academia or a small company can do. This is the beauty of our industry and our field.

We like to see our people challenge existing doctrine and hypothesize new ones, after all, isn't that how we are trained through grad school and postdoc training, but somehow and somewhere it starts to seem to difficult to do, especially when you try to get a paper accepted or a grant rated among the top 10%. We don’t want to lose our edge, even if we have to learn to better place it. We will continue to move this way, and we want you to come along for the ride.

Put your fluorescence in perspective and know your true RT-PCR results with these Specialty Oligos from Allele Biotech!

Allele is now distributing to its customers the newest technology in RT-PCR published less than two weeks ago. It is a RT PCR probe that utilizes fluorescence resonance energy transfer (FRET) and TaqMan methods in order to provide an internal positive control (IPC) for real-time PCR assays. This new RT-PCR method consists of forward and reverse primers, a regular TaqMan probe, and the new triple-labeled FRET-TaqMan Probe affixed to a plasmid, which negates the uncertainty in the presence and effects of amplification inhibitors that could generate a false negative in your experiments. Two emission sources are all that are required for the FAM and Cy5.5 fluorescence signals at 530 nm and 705 nm respectively. You do not need color compensation software, multiple excitation sources, or even a high tech laser to initiate excitation with this system.

If your RT PCR instrument only has one light source to excite the fluorophore then the FRET-TaqMan method is the only way to exploit the IPC technology. Fortunately Allele Biotech is selling these triple-labeled probes at a great price! Put your fluorescence in perspective and know your true RT-PCR results with these Specialty Oligos from Allele Biotech!

What will be the outcome?

Nobody knows for sure of course, but the number of demonstrators on the street can give Iranian demonstrators unfounded tactical optimism as I felt 20 years ago on Tian'anmen square. No matter how many million people are out on the street, and how determined they are to fight to the end, as the citizens of Beijing were back then, if the government wants to use real force, there will be completely unbalanced unsustainable fight. If it comes to using tanks and machine guns, the only chance will be somehow most of the troops are convinced that shooting or running over unarmed people is an order that can absolutely NOT be carried out.

After seeing Tian'anmen square, military personnel during many mass demonstrations in 1989 Eastern European countries like Romania felt that what they saw on TV a few months ago should not happen in their own country in any case. They'd rather shoot the dictator like in Romania. Maybe reminding everybody of the bloody and horrific street scenes of the Tian'anmen event will be helpful to prevent it from happen again, anywhere.

watching cnn, remembering 20 years ago

This time, from a distance. The show of number by people, the quite period of the power, then the characterization of the movement as against the country, and "western power backing" routine with no details(still no such foreign meddling was eventually shown by evidence 20 years after it was used as one of the reason for cracking down in Tain'anmen square), the escalation on the street.

Iranian people are brave, determined, and very smart. The rooftop shouting at night, the changing of tactics for demonstration, and continuation with communication channels clamped down and clear leaders missing, god I didn't think I'd see history of such scale happen in front of my eyes again in my life. Justice and people's rights will win in the end.

Allele Biotech: From one green revolution to another, Go Freedom in Iran!

The pictures of hundreds of thousands of people on the streets in Tehran eerily reminds me of what I experienced in Tian An Men square: the orderly conducts by the protesters, constant reminding each other to keep lines, maintain non-violent behavior, and obey whatever "orders" that come down from whoever that appeared to be the leaders of the group, as a whole or in small divisions. The crime rates in Beijing before the fateful day of 6.4.89 were at historical lows because citizens never felt that they were so responsible for everything that went on around them. Also similar between the then and now are the blocks on news media, and small incursions between protesters and police forces.

Soon after June 4th, 1989 came the wave of changes in Eastern Europe--people saw what happened in China and they wanted nothing like that in their own country. Hope that is the outcome for Iran.

Allele Will Bring a New Family of Fluorescent Proteins to the Market

Allele has signed an exclusive co-development and marketing agreement with the Swedish high tech company, Innoventus, to work with Dr. Olle Israelsson of the Karolinska Institutet on a novel class of fluorescent proteins.
These proteins were discovered in Amphioxus, a type of small fish that can be found in beach sand, which is believed to be a very primitive cordate species. Compared to jellyfish and coral, from which virtually all of the currently used fluorescent proteins were isolated, Amphoixus are closer to mammalians and their proteins may find great application in human cells and other commonly used animal models. In addition, there are a large number of protein variants that can provide different spectra and other important physical properties such as photostability and photoconvertability.
Allele Biotech’s plan is to first introduce several new fluorescent proteins of different colors to the market as immediate alternatives for fluorescent protein customers. The next step is to continue to evolve and mature these proteins to create more advanced proteins with desired properties suitable for live animal imaging or more advanced applications such as PALM/STORM and SIM. Allele Biotech has on its team of fluorescent protein research staff and collaborators, some of the most highly regarded scientists. With these resources, Allele Biotech plans on committing to long-term development of truly user-friendly fluorescence imaging products.
These new class of fluorescent proteins will be integrated into Allele Biotech’s current products including: retro/lentiviral vectors, baculovirus and bacmam systems, as well as iPSC and RNAi constructs.

Time to renew the SBIR law and the fight is on again.

The following information is courtesy of Rick Shindell
at SBIR Gateway, we are passing it along to make those concerned to become aware.

The four House bills were marked up and approved on June 11, 2009 by the House Small Business Committee's Subcommittee on Contracting and Technology and should go to the full SBC committee next week. The Senate bill is scheduled for markup June 18, 2009.


SENATE SBIR/STTR REAUTHORIZATION BILL S.1233 The Senate's SBIR reauthorization bill was introduced June 10, 2009 and sponsored by SBE committee chair, Mary Landrieu (D-LA), and ranking member Olympia Snowe (R-ME).

At the time of this writing the bill was not yet available from the government printing office, so we can't give you a link to it. We can provide you with an overview. It is close to but not exactly the same as last year.

Important points include:
* Extension of termination dates - 2023 (14 years)
* Improvements to strengthening the SBA Office of Technology
* Increase SBIR allocation by 0.1% per year (starting in FY-2011) until reaching 3.5% in FY-2020
* Increase STTR allocation to .4% for FY-2011; .5% for FY-2013; 0.6% for FY-2015
* Increase SBIR/STTR award levels to $150k phase I and $1M for phase II
* Awards shall not exceed 50% above recommended award levels
* Elimination of Phase II "invitation" process (i.e., DoD)
* VC small biz eligibility compromise limited to 18% of NIH SBIR Award funding, 8% at the other 10 agencies
* Allow small business to partner with federal labs or FFRDC without requiring a wavier from SBA
* Reinstate State and Rural outreach programs
* SBIR STEM Workforce Development Grant Pilot Program
* Continuation of Commercialization Pilot Program (DoD)
* Establish Commercialization Pilot Program for civilian agencies
* Nanotechnology Initiative
* Accelerating Cures - NIH Pilot
* Accuracy In Funding Base Calculations (keep em honest in the 2.5% extramural calculations)
* Increase in technical assistance from $4k to $5k
* SBIR and STTR Special Acquisition Preference

It is highly recommended that if you like the basis of this bill, contact your Senators and ask them to cosponsor this legislation, (S.1233 - A bill to reauthorize and improve the SBIR and STTR programs and for other purposes). This is very important if you want the Senate version to stand a chance on passing.

A tidbit you might have already known, the Challenge Grant through NIH’s ARRA stimulus program received 20k applications for some 200 to 400 awards.

The NIH stimulus grants do not have the SBIR obligations by a last minute change. How may all these affect Allele’s operations? We have submitted 3 grants to the NIH in the last 3 months, with total 4 now pending. It means that we sure are interested in NIH funding, which was, after all, how our company was started. On the other hand, we are also glad that we do have ongoing sales and services that link us directly to users of our technologies. In the current difficult economy and tight funding environment, we strive to be a company that supplies most essential biological research tools that could save average labs some 20-50% cost per item compared to buying from companies like Life Technologies and Clontech, etc. At the same time, we want to provide the convenience to our customers by covering a sufficient number of common reagent areas, a value small specialty companies normally do not offer. See our next blog for more comments on being a flexible and able provider of everything essential.

从大学毕业到毕业十年再到二十年，我思想上最大的变化是什么？（一）

大学毕业后，由于6.4的影响太大、太近、也太突然，我虽然把它当作第一大事向每一个有机会交谈的人谈起，也从没有怀疑过谁是正义一方谁是邪恶一方，但并不能把这一基本的对错观与自己为人处事、安身立命的思想根基联系起来。我的人生的骄傲和支点，或者说我之所以是我和愿意继续是我的原因，还在很大程度上来自于和其他团体、组织、或个人的“privileged” 关联。第一种表现可能就是在向不熟悉自己的人介绍自己的时候会说起自己的大学、中学、甚至小学，不是作为了找寻他乡遇故知的惊喜，而是象动物交手前炸炸毛、亮亮翅以确立自己在每人心目中的地位。如果没提这些则多半儿是不好意思太欺负人，在确认对方“不是个儿”（no match）之后。在听到来往的人大谈“我认识的谁谁谁多么优秀”从而让你感到她/他也如何优秀时，也只是轻微地诧异和有不适感—物以类聚人以群分？至于说到种族、国家背景之类，则是一种脱离政治的潜意识性的反应，即说我国家不好便是在说全国人包括我生活在中国的父老乡亲，这也必然延伸到所有中国种的人或叫华人，包括自己。这种感觉一直延续到毕业后十年左右。

回想起来，这里面还有两个稍微深层的原因。 1）初到美国的不适应感和骤然失去许多以前惯有优势的失落，比如在中国多少年来享受的是北京人、说标准普通话、甚至汉族的正宗、正统的优越感随着初说英文、在美国没有了家庭背景和任何经济支持、变成少数民族等荡然无存。2）受70年代出身论和80年代变种而来的精英论影响，觉得或是希望社会的资源和人们的尊敬应该是按某种已经确立过的结构确定并分配下来。因为我是北大的，所以我已经证明了自己在这一结构中的地位了，没必要请不要打破。记得那时候申请学校的申请表中经常会写“本人来自中国数一数二的大学，它们的录取万里挑一、十万百万里挑一”言下之意凭这个Harvard、Yale都比不上，此时不录取我更待何时。另一个表现就是刚到美国的第一年里凡是自命不凡的命校毕业生的主要精力都花在了转学到“更好”的学校去的热潮中。其结果之一就是反而很多在中国非名校毕业生反而踏踏实实作试验，然后走学术路线开实验室教别人做试验。这里没有对任何职业有任何褒贬的意思。通看全文你就会体会调揩是对所有人和事。

待续

Four ways to clone your PCR products, which one should you use?

Cloning of a PCR fragment is typically achieved by one of the following methods:

1) TOPO cloning using a TOPO blunt vector for PCR products generated with Pfu or many other high fidelity thermostable DNA polymerases, or TOPO TA vector for PCR made with Taq. The efficiency is high and so is the cost partially due to the exclusivity of Life Technologies (Invitrogen) to offer topoisomerase-based cloning vectors. TOPO kits always include competent cells, controls, etc. with the topoisomerase-conjugated vector, further increasing the prices to about $25/reaction. The background of TOPO cloning is normally low as the enzyme-linked ends of the linear vector DNA do not allow self-ligation; however, occasionally circular plasmid formation proceeds through recombining one end of the linear vector to a vector sequence hundreds of bases downstream of the other free end.

2) Restriction digesting PCR fragment for ligating to cloning vector. This method allows you to have the freedom to choose virtually any plasmid vector that immediately suites your experiments intended for using the clone. Unfortunately the efficiency of cutting restriction sites introduced to the ends of linear PCR DNA pieces is often extremely low, even with extra bases added to the outside of the restriction enzyme sites. Background ligation by vector self-ligation or erroneous ligation to contaminated DNA becomes prominent when properly cut insert DNA is not present in sufficient concentration due to poor restriction digestion. The cost should be the lowest using this method, given that you don’t have to do repeated digestion with increasing amount of restriction enzymes and ligation with batch after batch of ligase.

3) End fusion via recombination assisted by end-chewing DNA polymerase or other DNA enzymes. The most actively promoted commercial product for this method is the In-Fusion line from Clontech (Takara). The cost of using the In-Fusion kits is somewhat lower than the TOPO kits, still above $12/reaction in most cases even without accompanying competent cells. At least there is an option of just buying the kit without competent cells, which is basically some dried-down viral DNA polymerase in separate tubes. Because in theory enzyme-assisted recombination can occur between any homologous sequences at the ends of linear DNA, the user has freedom to choose any vector. In practice however, it is questionable how much the added DNA polymerase actually help the efficiency since in our hands we found sometime the no-enzyme controls worked better than using the supplied enzyme. Furthermore, we also observed vector dependence of cloning efficiency.

Preannouncement: Allele Biotech will soon offer a linear form of DNA for both entry point PCR cloning (i.e. putting linear PCR product into a circular vector so that subsequent restriction digestion at the ends of the PCR fragment will be easy) and basic bacterial protein expression. There will be no enzyme required at all and the cost will be much lower than similar products from other suppliers.

4) TA cloning is a simple method of cloning DNA fragments created by a PCR reaction catalyzed by the Taq DNA polymerase. The PCR product with 3’ overhanging A base is ligated to a linear vector with overhanging 5’ T. This method was devised after the discovery that the 3’ ends of PCR products generated by Taq polymerase contain an unmatched A base added by the terminal transferase activity of Taq polymerase. The T/A matching so provided helps increase the ligation efficiency over blunt end ligation such as ligation between Pfu-generated PCR and vectors cut by EcoR V or Sma I. Therefore, even when high fidelity enzymes are used for PCR, many researchers choose to add the extra A base by incubating the PCR product in the presence of Taq for about 15 min. Even though a linear vector with a 5’ overhanging T is not trivial to produce, the cost of TA cloning vectors is still much lower than TOPO or end-fusing vectors. Allele Biotech’s just-launched TA cloning vector also utilizes a LacZ-based blue/white selection. The background ligation rate is very low while ligation with insert typically results in a few hundred colonies by standard procedures using Allele’s Extreme Efficiency competent cells.