Fluorescent Protein-Based Assay Development

This blog is a preview of what is to be launched as a new Service Group. Allele Biotech is restructuring its CRO capabilities in the assay development area by combining its fast expanding fluorescent protein portfolio, viral vector and packaging expertise, as well as newly granted patents in shRNA. The focus of this post is fluorescent protein in biosensor and screening assays. A modified version will be used as the landing page for the FB-Based Assay Development Service.

Overview:

Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.

Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.

FPs as fusion tags:

Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.

FPs as expression reporters:

FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.

New Product of the Week 03-08-10 to 03-14-10: mWasabi 2A or IRES dual expression vectors (http://www.allelebiotech.com/shopcart/index.php?c=216&sc=34) ABP-FP-W2A10, orWIRES10

Promotion of the Week 03-08-10 to 03-14-10: for a limited time on Thursday, to be announced on our Facebook page (http://www.facebook.com/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957#!/allele.biotech?ref=profile), a strikingly low price will be honored for a commonly used lab reagent or equipment. This is the second week of the follow-us-to-the-basement promotion.

Tags: assay development, biosensor, cell based assays, chemical screening, circular permutation, fluorescent protein, FRET, GFP, mTFP1, mWasabi, promoter-FP, RNAi screening

Oligo Giveaway Results are in!

Last week our 10th Anniversary Free Month of Oligos Giveaway contest ended with the selection of winner Allan Kelleher from the University of California San Diego. Our contest was exclusive to our Facebook friends and fans but we announced the giveaway and updated the status of the contest via our other social networking channels. I got the opportunity to meet with Alan this last week at UCSD’s Bonner Hall where he is hard at work in molecular biology research.

We gave him a certificate to confirm his win and took a few photos. He was a really nice guy and we are happy that our $1600 worth of free oligos will assist him in his experiments. I will post the pictures on our social sites soon.

I was really eager to promote this contest. At Allele we wanted to do something that marked our ten years in business in an impressive way while at the same time abiding by our philosophy of encouraging the advancement of research. Oligos were an organic choice as they were our first commercial product on top of being a universally useful tool in the industry, which is evident in their use by medical researchers, molecular biologists, physicists, oceanographers; the list goes on.

To hold the contest exclusive to our Allele on Facebook friend/fan base was another organic choice as most of our contacts on this format are bench researchers. Some are just fans that are interested in our weekly product promotions and new product announcements. However, most of our Allele on Facebook contacts are friends. This allows us to see their bio questions. In frustration they will post, “Has anyone had experience with this type of ligation…?” Facebook gives our friends the opportunity to get help from top scientists for free and often during their workday for the fastest access to information available.

For all the benefits available to you, join Allele’s friends on Facebook today and be the first to know about everything Allele Biotech has to offer!

Tags: allele on facebook, FREE oligos, new products, ontest results, weekly promotions

How I started my company and why–Inaugural Event by San Diego Entrepreneurs Exchange (SDEE)

For current graduate students, postdocs, and holders of other “in-transient” positions in bioscience-related fields today, a persistently resounding question on our minds is “What path should I follow at the end of a long and ragged journey of training?” Interestingly in our industry, like downhill skiing you see in the Winter Olympics, once you start one path it is not an easy switch to get on another.

Many of the Ph.D.s in biomed share the general view that an independent research position typically at an academic institute or non-profit organization such as San Diego’s local Salk, Scripps, or Sanford—Burnham, is the goal of the many years of training. Others soon realize that there are numerous research jobs at biotechnology and pharmaceutical companies that will make good use of their expertise, experience, and unique background knowledge in a particular field. And of course there are those who “defect” to different industries that may or may not directly relate to their extensive experience in wet labs, such as working in intellectual property laws, clinical trial management, biomedical sales, business development and management.

Research in major pharmaceutical companies (big pharma) normally focuses on a project with set goals, milestones, and layers of monitoring and management. That is how a large team can function together and get the tasks done in a timely manner. Working in smaller biotech companies can be much more flexible, researcher-initiated, and in many ways fun. On the other hand, you will be required to do much more than reading papers, designing experiments, obtaining and interpreting results. Starting a small biotech company is by no means an easy path to take, but if done correctly with some luck and a lot of determination, it can be a very rewarding career. You will get to utilize to the maximum extent of all your intelligence, knowledge, vision, and personal relations. You also have the opportunity to do real cutting-edge research in various areas, and see the fruits in journal publications, grant awards, as well as in the wild wide market.

The San Diego Entrepreneurs Exchange (SDEE) was founded by local San Diego entrepreneurs in order to provide a voice for the early stage technology startup, to encourage new entrepreneurs, and to sponsor networking and educational events that help develop the skills necessary to bring funding and business to the San Diego area.

The inaugural SDEE event to be held Wednesday March 10th at 5pm. It will help answer some of the questions you may have been thinking about regarding starting or working in a startup biotech company. Allele Biotech’s founder and CEO Dr. Jiwu Wang will be among the speakers. Ten years ago Dr, Wang was a postdoc at UCSD with an NIH fellowship, right before he started Allele with a number of NIH small business innovative research grants. He will talk about the ultimate “academic freedom”–doing any research you want but completely at your own risk– as the reason to start a technology-focused company, and the lessons he learned the hard way about running a lab vs organizing a business. Other speakers include CEOs from a number of San Diego biotech companies with great stories to share with postdocs and others. The talks will be brief yet informative, and on-site interactions are encouraged. The Sanford-Burnham building 12 is outside the main campus, with plenty of free parking. Click here for more details about the event. http://www.allelebiotech.com/allele3/SDEE-First-Event-Announcement.pdf (at AlleleNews). Let us know if you are coming by emailing to events@sdentrepreneurs.org

New Product/Service of the Week 02-15-10 to 02-21-10: Viral shRNA design and packaging services, packaging 2ml virus at 10e8 TU/ml for less than $1,400.

Promotion of the Week 02-15-10 to 02-21-10: FREE spreading beads (ABP-CE-CCCSB100, 500) to go with any competent cell order.

Tags: biotech, biotech careers, biotech jobs, biotech startup, pharmaceutical companies, postdoc jobs, research positions., San Diego Entrepreneurs Exchange, SDEE

Allele Received Broad Patent on DNA-Expressed RNAi in China

Allele Biotechnology & Pharmaceuticals, a San Diego based private company with associate offices and laboratories in China and distribution channels in 30 countries, was granted a major landmark patent in China in the field of RNA interference (RNAi). The patent CN02828345.7, issued on January 20, 2010, covers compositions of DNA molecules that can be transcribed into RNAi-mediating RNA molecules, including the commonly used shRNA and miRNA-like designs. The patent also grants Allele Biotech rights to the process of introducing such DNA molecules into cells. To induce gene silencing by RNA interference, researchers often bring DNA molecules that encode interfering RNAs into cells via plasmid or viral vectors. The rights to use related technologies for the purposes of completely or partially abolishing gene functions through the mechanism of RNAi are granted to Allele Biotech.

Additional claims include methods of studying gene functions using DNA-encoded RNAi agents, or modifying gene expression profile by introducing gene expression-altering DNA molecules that will induce RNAi. The patent further protects the use of DNA-mediated RNAi in creating cell, animal models, and for curing human diseases. According to a Nov 2009 CreditSuisse analysis on the pharmaceutical market in China (and a number of other reports by JP Morgan as well as Morgan Stanley research, etc.), the drug market in China will double by 2015 and the expected revenues for major pharmaceutical companies are in the billion US dollar range each. Many large drug developers have opened research centers in China. For instance, Novartis just announced a 1.25 billion US dollar investment in Chinese R&D centers, making Shanghai one of its top three global research centers. Roche, Pfizer, JNJ, AZN, Bayer, and LLY also have substantial investments in R&D there. Some of their research teams have plans to use the virus-carried shRNA technologies in oncology and other areas, either as screening/validation tools or as therapeutic candidates. Such activities in China are now under the Allele’s recently granted RNAi patent.

The Contract Research Organization (CRO) industry in Shanghai, Suzhou, and Beijing has seen significant growth in the past few years, benefiting from R&D cost cutting in Western countries and the flow of Western-trained researchers back into China. The focus of the CRO business also shifted from chemical synthesis towards one-stop service, including functional screening and animal testing. The clarification of the RNAi patent landscape by the current granting should make the relevant CRO applications of RNAi more mature. It should also provide both the service and the customer companies with a clear route to licensing and/or collaboration.

Most major biomedical research tool and reagent companies have established themselves in the Chinese market and seen fast-growing revenues due to large funding increases to biomedical research in China. For example, Life Technologies, Promega, Millipore, Thermo Scientific, and Sigma-Aldrich all sell RNAi kits that use DNA template for expressing shRNA in mammalian cells, either by viral infection or DNA transfection. In addition, there are many local companies in China that provide reagent kits as well as services.

The Allele patent specifically states claims on reagent kits that contain shRNA-encoding DNA molecules. While being the first in China’s RNAi market, Allele Biotech manufactures in the United States and sells world-wide a set of RNAi kits in the form of retroviral or lentiviral vectors, plasmids, and linear DNA—all of which have superior design for precise shRNA production. As a matter of fact, Allele Biotech helped introduce the RNAi concept through a series of workshops in major universities in China for 3 consecutive years since 2002, at a time when most biologists had just heard of RNAi.

Allele Biotech intends to fully realize the value of this broad patent by providing opportunities to R&D centers, service providers, and reagent sellers to license at reasonable fees, so that this great technology will continue to be widely used and further developed through original research and investment. Allele Biotech intends to set licensing fees on a sliding scale in several aspects:
–the closer a drug gets to market, the higher the fees;
–the smaller the company, the lower the fees;
–the earlier the license is negotiated within an industry sector, the lower the fees.
Allele’s attorneys in China have already been contacted to start drafting plans for licensing deals and patent rights execution. “While stressing wide access, limiting the number of licenses in China is not completely out of the question. In general we want to grant all-application, non-exclusive, low-cost licenses to many companies to keep the costs affordable.” says Dr. Jiwu Wang, Allele’s CEO and the inventor of the patents. “However, if a dominant player in a particular application area is more interested in some exclusivity, a co-exclusive or conditional exclusive license may be negotiated”.

A brief background about RNAi patents:
–The original Fire and Mello patent claimed double-stranded RNAs longer than 25, eliminating use in most mammalian cells.
–The few other RNAi patents granted in the US, Europe, Japan and other markets so far mostly concern chemically synthesized siRNAs.
–The Tuschl I and II patents, with the latter being frequently mentioned in the news because it has generated hundreds of millions of dollars in licensing fees, concern siRNAs suitable for mammalian cells, but they are either chemically synthesized or processed in cell lysate.
–The Allele patent family includes 3 issued US patents on using RNA polymerase III promoter (e.g., commonly used U6 promoter) for generating RNAi. The core of the Allele patents describes making siRNAs that can be of 19 to 25 basepairs long, which are not covered by the Fire and Mello patent. Further, these transcribed siRNA are not chemically synthesized; therefore, they do not conflict with the Tuschl patents. The Allele patent in China has an even broader field of granted rights, covering any DNA-based gene silencing using double-stranded RNA as intermediates.

New Product of the week 020810-021410: Ready-to-use retroviral particles expressing mWasabi, the brightest GFP, ABP-SC-VIREMW1 or 5.

Promotion of the week 020810-021410: Ready-to-use lentiviral particles expressing hNanog and hLin28, ABP-SC-LVINANO and ABP-SC-LVILN28,high titer, 50% off list price, promocode nglnkrm0210.

Tags: Alnylam patent, Benitec, gene silencing, ISIS patents, Max Planck, miRNA, RNAi, RNAi patent, shRNA, siRNA, Tuschl patent, UMass Ownership

Submit Your Pictures and Enter to Win $100 in Cash

Allele Biotech is holding another fun contest to give back to our loyal supporters and everyone has the chance to win $100 in cash! We are looking for interesting and relevant pictures, illustrations, and computer generated art that pertains to any Allele Biotech Product or service to replace the flashes on our homepage. If it is chosen to be used we will give you $100 cash!

Check out our technical data sheets, read our past blogs, and even use your own lab experience and impressions with Allele Biotech Products to come up with your entry. There are over 1000 products to choose from so everyone should have lots of inspiration!

All you have to do to enter is submit your picture to our facebook inbox! The picture has to be of your own creation and/or one to which you own exclusive rights. Response time and prizes will take about a week.

Let your creative juices flow and you could get $100 in return. Your picture could be funny, serious, or even super nerdy!

Used to promote our Sapphire(TM) Baculovirus Expression Service

All photos, illustrations, and computer generated graphics (aka. “the picture”) submitted must be lawfully owned by entrants who submitted them. Pictures that are not chosen will not be used in any way by Allele Biotech. Winners may collect their prize via cash, check, or a one-time Allele credit of $100 good toward any Allele Biotech purchase. Allele Biotech has full rights to pictures selected for use in Allele advertising and winners must forfeit any future rights to the picture. Contest to run indefinitely and to be terminated at any time.

Tags: cash prize, contest, submit pics for cash, win $100