10 Years of Allele Biotech

Facts about Allele’s 10 years in business:

Products

New product lines added in 2009: iPS cells, Camelid Antibodies, DNA synthesis chemicals, Recombinant Proteins

Highlights: HiTiter Lentiviral Systems, Baculovirus for Mammalian Expression (BacMam), Feeder Cells, shRNA on Viral Vectors, shRNA Validation FP Vector, ProperFold Protein Folding Vector, Validated AllHPLC synthetic siRNA

New Service Groups in 2009: Viral Packaging, RNAi Validation/Screening, FP-based Assay Development

Numbers

Since April, we have added at least one new product every week! We currently run one new promotion per week as well.

A bit of history–did you know that…

Allele Biotech obtained 5 NIH grants in its first three years since establishment. As a matter of fact, Allele Biotech was funded entirely by NIH grants

Allele filed its first patent application in its second year of operation, which was on DNA-driven RNAi and resulted in an outlicensing deal with Promega. As result of the applications, Allele has received 3 US patents on DNA-encoded shRNA, siRNA using promoters such as U6 and H1.

During the past 10 years, Allele was the first to sell U6-based RNAi vectors, the only supplier of bFGF-expressing feeder cells for iPSC, most likely a top 3 provider of baculovirus expression systems, camelid antibody products, iPS creating viral particles, and the most active commercial developer of fluorescent proteins.

Tags: 10 years in business, Allele Anniversary, camelid antibodies, NIH grants, oligos, RNAi

GPCR: problems and resolutions in hihg content screening

A review in the December, 2009 issue of Nature Methods, “GPCR: insane in the membrane” by Michael Eisenstein impressed me greatly. It presents a clear and wide view of the GPCR research fields, from the basic background knowledge, to difficulties and current solutions in real cases, to future development. After careful reading and thinking, I felt that there were still several questions left to for me to pounder.

1) The Plot Thickens

“In many cases, you identify a GPCR as a target based on physiological data, and the receptor might be expressed in the brain in a particular neuron, but then you perform a high-throughput screen in over expression, immortalized cell lines that are nothing like the cell in which the receptor normally resides,”This is the inherent problem for all the engineered and artificial cell based assays most widely used in GPCR research right now. As stated in this review, “There are so many ways to be misled by using an over-expressed receptor in a non-native cell line”, it was shocking to me, making me realize that cell line screening does not only have a problem of getting raw data of low quality, but also a problem of being seriously misleading, a problem of direction rather than efficiency.

Directed differentiation of iPS cells and ESCs might be one of the potential solutions to this problem. However, from our experience in iPS cells dedifferentiation and differentiation at Allele Biotech, as a main vendor of iPS cell reagents, it is hard to make this platform in current situation suitable for high content analysis of GPCR. As much as we wish and believe, like many other researchers and the general society, that stem cells will contribute significantly also to the drug screening field, much work needs to be done before that happens.

Primary cells could be a good alternative. Based on data from Allele Biotech’s trade partners in the primary cell business, the primary cells market has been expanding dramatically since late last year. I believe that the utility and advantage of primary cells have been increasingly appreciated. However, there are still some problems with using primary cells in GPCR studies. For example, neurons are hard to obtain and can not amplify in vitro, in which cases there seems to be no chance to satisfy the need by screening. In fact, neuron research is one of the main GPCR research fields. Therefore, this is a must-fix problem.

How about “Immortalized Primary Cells”? The immortalized primary cells maintain the properties of primary cells well, relative to established cell lines. We have built our own advanced immortalization technology, based on which new products and services will be released in the first season of next year. During the last several years, we have successfully immortalized immunological cells and cancer cells. WE have yet to have the experience of working with neurons. Anyone who has interests of cell immortalization is welcome to contact us for collaboration or custom service. “Introducing Cost Effectiveness to your research”, just as the slogan of Allele Biotech, we will be proud to serve you with cutting-edge technology and cost effectiveness!

2) Without a mark to be continued…

By Niels Yuhui Ni, researcher at Allelle Biotech

Allele Biotech is 10 Years Old and Celebrating with FREE Oligos!

Allele Biotech is 10 years old!!!!!!! December 1999 was when Dr. Jiwu Wang and colleagues started this great company as a DNA oligo and siRNA service provider to the San Diego area. Since then he has fearlessly lead Allele into the forefront of the biotechnology industry with multiple RNAi patents, numerous NIH grants, revolutionary iPSC and fluorescent protein technologies, the acquisition of Orbigen, a continuously growing catalog of over 1000 molecular biology products and signature Allele Biotech Reagents, and a business culture that is approachable, encouraging, and reverential of research advancement through global communication and collaboration.

What a difference a decade makes. Today Allele Biotech is a top oligo service provider all around the country with customers in all of America’s major academic institutions. Currently, we are the sole oligo provider on the University of California San Diego’s central purchasing site, Marketplace; a collaboration designed to provide top quality oligonucleotides to UCSD research departments while saving them thousands of dollars annually. We now produce many of our own oligo synthesis and modification reagents, further cutting the costs to our valuable customers which enabled us to continue operations this last year without raising prices due to the worldwide acetonitrile shortage which more than quadrupled in cost! Allele Biotech has stood out over the years not only to our loyal customers but other oligo providers as well. Less than 5 years into our operation we were approached by one of the well known, top three, oligo providers in an attempt to buy us out! We resisted and are still here to proudly serve the research community with the Allele brand!

It all started with oligos…Now ten years later we want to honor our accomplishment by giving away a FREE month of oligos to one lucky customer! To enter you must be an Allele Biotech Facebook fan or friend. A winner will be randomly selected from our friends/fan pool on Sunday, February 14th, 1210 at noon. That lucky winner will receive FREE oligos for the month of March 2010! Limitations apply. Click here for terms and conditions.

Tags: 10 year with Allele Biotech, 10th anniversary promotion, anniversary promotion, FREE oligos, oligo services

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.

Tags: cell imaging, CFP, FLIM-FRET, Fluorescent proteins, FP, FRET, FRET acceptor, FRET donor, FRET pair, GFP, GFP fusion, mTFP1, mWasabi, subcellular localization

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

Liang Yin, Jiwu Wang, Lung-Ji Chang and Yichen Wang

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.

Flow cytometry analysis of reprogrammed human marrow stromal cells.