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 Biotech Spotlight Promo for ASCB Dec 09 Meeting!

This year our President and CEO, Dr. Jiwu Wang Ph.D., will be presenting at the American Society for Cell Biology meeting in San Diego, December 5th through 9th. Dr. Wang will be presenting results of two studies that involved the Allele Biotech Fluorescent Proteins and iPSC product lines:

Monomeric photoconvertable fluorescent protein variants produced by directed evolution for brightness and efficient photoconversion – a collaborative effort with the Campbell lab at the University of Alberta

Increased efficiency and speed of reprogramming of human cells into induced stem cells using high-titer lentiviral vectors encoding cell cycle progression and survival genes – a collaborative effort with the Chang lab at the University of Florida

In honor of this prestigious occasion Allele Biotech is having a Spotlight Promotion on all Fluorescent Protein and iPSC Products! The promotions, which will vary from product to product, will include 10% and 20% off price reductions, FREE shipping, and even “Buy 2 get one Free” deals!

Products eligible for the Spotlight Promotions begin with:

ABP-FP-____ Catalog

ABP-SC-____ Catalog

To qualify for these promotions you must be attending the ASCB meeting in San Diego and provide us with a copy of your registration form or be one of our loyal facebook, twitter, or myspace friends. Any questions can go to oligo@allelebiotech.com

Call for details and ask for info on the Spotlight Promotions! Offers good now through December, 9th 2009!

New Product of the Month 11/23-29/09: ThermoExp500 PCR machine (thermocycler) $4,250.00, with almost twice as fast temperature ramping as MJ’s TC1000, and more reliability.

Tags: Allele Biotech Promotions, Allele Biotech Sales, American Society for Cell Biology, ASCB San Diego 2009, fluorescent proteins discount, free shipping, iPSC discount, MJ research, PCR machine, TC1000, thermocycler

RNAi Design, Validation and Target Screening

Since Tuschl et al. published the first empirical guidelines on how to design effective siRNA [1], the most significant advancement (based on the understanding of biochemical mechanisms of RNAi such as how RISC is assembled) is the recognition of asymmetric thermostability of the 5’ end of the antisense strand (AS) relative to that of the sense strand (SS) [2, 3]. siRNAs with an A/T-rich AS 5’ end can be more easily integrated into RISC. By biasing against the sense strand for RISC loading, the off-target effects due to the presence of the SS (as one of the sources of off-targets effects) can also be minimized. In recent years datasets of increased number of siRNAs and shRNAs became available and statistical analysis suggested additional rules for RNAi design. These newer rules in general define the siRNA prediction parameters in more detail, for instance, the number of bases of the 5’ ends that should be included when calculating asymmetric thermostability, base preferences at each particular position, and the identity of the 2 nt 3’ overhang [4, 5]. Computer programs and websites are developed based on these features also resulting from NIH funded research through universities and organizations. Among the well-known ones, Design of SIRna (DSIR at biodev.extra.cea.fr/DSIR/DSIR.html) and the shRNA search program at the Broad Institute (broadinstitute.org/genome_bio/trc/publicSearchForHairpinsForm.php) are freely available.

Several companies such as Open Biosystems, System Biosciences, Dharmacon/ThermoFisher, Sigma-Aldrich, Invitrogen/LifeTech, provide premade RNAi reagents against various numbers of human and rodent genes. Although some product lines from these suppliers are labeled as validated RNAi reagents, apparently only one reveals clone sequences and only a few hundred among the claimed 4,500 shRNA clones. It is not possible to find what shRNAs are used against any target gene from most companies even though many of them claim to have a few hundred pre-validated constructs. Some of them may provide additional information upon purchase.

Even with the recent advancement of RNAi design technologies, prediction of effective RNAi is still far from accurate. Depending on the datasets used to score the success rates of the programs at DSIR, Broad or any other software, the general consensus is that about 50% of predicted RNAi target sequences will be effective, resulting in better than 70% gene knockdown. Allele Biotech uses a software that was trained with known RNAi results to predict siRNA target candidates on a given mRNA, and then applies an additional set of rules to pick the most promising candidates. Off-target effects caused by partial-matching between AS strand and untended targets are reduced by searching the chosen site against the NCBI gene base. The basic rules Allele Biotech uses include most currently known ones and are similar to what are listed by The RNAi Consortium (TRC) program at the Broad Institute.

Criteria for RNAi design:
(1) Overall GC content is between 30-55%
(2) The 4 bases at the 5’ of AS is more AT-rich than those of the SS
(3) The first base of AS and SS 5’ is preferably A/T and G/C, respectively
(4) “U” is preferred at the 10th position of the antisense from the 5’ end
(5) “C” is to be avoided as the last base of an overhang
(6) Avoid 4-nt mono-nucleotide regions
(7) Avoid 6-nt GC-rich regions
(8) If possible, do not include those with apparent secondary structures

These selected rules are based on a number of publications (for example, [4-6]), but it is impossible to include all known rules, many of which conflict with each other. In case of conflicting rules we rely more on recent discoveries and our own experience from providing RNAi service during the past 8 years.

Allele Biotech provides RNAi validation and screening services to customers using synthetic siRNA, linear DNA cassettes with engineered Pol III promoter, and shRNA expressing lentiviral vectors in high throughput formats. In a unique design, all RNAi target candidate sequences of a gene transcript are fused consecutively to a bright green fluorescent protein, mWasabi, on a lentiviral vector. Instead of analyzing gene silencing by QPCR, the initial selection of effective RNAi can be performed by measuring fluorescence.

RNAi screening has been conducted to identify correlations between gene functions and cellular phenotypes such as synthetic lethality among DNA damage signaling and repair pathway factors. Successfully performing high throughput screenings requires capabilities of efficient RNAi design, viral packaging, fluorescent proteins, and advanced cell culture and analysis techniques. In addition to these capabilities, Allele’s RNAi services are provided with access to commercial use of Allele’s own patents on Pol III promoter driven shRNA expression, and licensed patents on lentiviral vector, packaging, and fluorescent proteins.

New Product/Service of week Nov 16-22, 09:

RNAi validation/screening service.

1. Tuschl, T., P.D. Zamore, R. Lehmann, D.P. Bartel, and P.A. Sharp, Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999. 13(24): p. 3191-7.
2. Khvorova, A., A. Reynolds, and S.D. Jayasena, Functional siRNAs and miRNAs exhibit strand bias. Cell, 2003. 115(2): p. 209-16.
3. Schwarz, D.S., G. Hutvagner, T. Du, Z. Xu, N. Aronin, and P.D. Zamore, Asymmetry in the assembly of the RNAi enzyme complex. Cell, 2003. 115(2): p. 199-208.
4. Vert, J.P., N. Foveau, C. Lajaunie, and Y. Vandenbrouck, An accurate and interpretable model for siRNA efficacy prediction. BMC Bioinformatics, 2006. 7: p. 520.
5. Zhou, H. and X. Zeng, Energy profile and secondary structure impact shRNA efficacy. BMC Genomics, 2009. 10 Suppl 1: p. S9.
6. Ui-Tei, K., Y. Naito, F. Takahashi, T. Haraguchi, H. Ohki-Hamazaki, A. Juni, R. Ueda, and K. Saigo, Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res, 2004. 32(3): p. 936-48.

Tags: cell based assays, DNA damage repair, gene knockdown, gene silencing, lentiviral vector, Pol III promoter, RNAi, RNAi design, RNAi screening, RNAi service, RNAi targets, shRNA, siRNA, Synthetic lethal

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.

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

The following information is courtesy of Rick Shindell
at SBIR Gateway, we post this excerpt to here to help more people who may be concerned to become aware of the situation.

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.

Tags: ARRA, GO grant, lab budget, lower costs, NIH challenge grant, NIH funding, research grants, research tools, SBIR