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.

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

AlleleNews- In pursuit of global information and communication

Allele’s ardent endeavor to create a platform for relevant, technical, biology news is paying off with the potential for everyone to benefit from our philosophy that all researchers should be given an opportunity to talk to the world about their discoveries while being able to learn about techniques they might otherwise have never heard of.

Our news site which provides the latest updates of Allele Biotech business and product developments is now also a platform for the global scientific community to introduce their techniques, products, and various other discoveries. While we still provide these news releases on our blog site and other social networking venues, we will utilize this multichannel approach for news announcements to cater to the assortment of appetites around the globe.

Our most recent addition to the “News Releases from Other Companies” section of the AlleleNews site is from Randox Laboratories announcing their development of a rapid, multi-marker screen for illegal drugs that requires as little as 7ul of urine for the screening of up to 10 drug classes. They postulate that this innovating technology will eliminate time and cost for high throughput laboratories at the rate of 1088 tests per hour!

We encourage all to contribute to and look to AlleleNews as an archetype for the exchange of cutting edge news from around the world. For submission approval please email oligo@allelebiotech.com with “AlleleNews Submission” in the subject line.

Tags: biotech news, cooperation in biotechnology, global science, lattest techniques, post your news, research around the world

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