Record number of papers citing the GFP-Trap group products in 2011

The following are references in regards to GFP Trap published in the second half of 2011 (not a complete list); a high quality GFP-binding protein based on a single domain antibody derived from Camelids. It is characterized by a small barrel shaped structure (13 KDa, 2.5nm X 4.5 nm) and a very high stability (stable up to 70°C, functional within 2M NaCl or 0.5% SDS). With much greater stability, specificity, and affnity, GFP-Trap®, the recent addition to antibodies for immunoprecipitation, should make GFP the most suitable tag for immunoprecipitation assays.

For live PubMed links, view this version please.

Krastev, D. B., Slabicki, M., et al. (2011). A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nature Cell Biology. 13: 809-818. PubMed

Aboobakar, E. F., Wang, X., et al. (2011). The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high temperature stress responses in Cryptococcus neoformans. Eukaryotic Cell. EC. 05148-05111v05141. PubMed

Uhrig, R. G. and Moorhead, G. B. G. (2011). Two ancient bacterial-like PPP family phosphatases from Arabidopsis thaliana are highly conserved plant proteins that possess unique properties. Plant Physiology. PubMed

Larance, M., Kirkwood, K. J., et al. (2011). Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway. Molecular & Cellular Proteomics. PubMed

Hattersley, N., Shen, L., et al. (2011). The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Molecular Biology of the Cell. 22: 78-90. PubMed

Rancz, E. A., Franks, K. M., et al. (2011). Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics. Nature Neuroscience. 14: 527-532. PubMed

Palmer, C. S., Osellame, L. D., et al. (2011). MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO reports. 12: 565-573. PubMed

Pichler, G., Wolf, P., et al. (2011). Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways. Journal of Cellular Biochemistry. 112: 2585-2593. PubMed

Mitchell, L., Lau, A., et al. (2011). Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4. PLoS One. 6: e25336. PubMed

Engeland, C. E., Oberwinkler, H., et al. (2011). The cellular protein Lyric interacts with HIV-1 Gag. Journal of virology. JVI. 00174-00111v00171. PubMed

Wang, C. and Youle, R. (2011). Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1′s inhibitory effect on Bak. Oncogene. PubMed

Tulloch, L. B., Howie, J., et al. (2011). The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation. Journal of Biological Chemistry. 286: 36020-36031. PubMed

Sun, L. and Wang, C. C. (2011). The Structural Basis of Localizing Polo-Like Kinase to the Flagellum Attachment Zone in Trypanosoma brucei. PLoS One. 6: e27303. PubMed

Bouttier, M., Saumet, A., et al. (2011). Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation. Nucleic acids research. PubMed

Matos, J., Blanco, M. G., et al. (2011). Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis. Cell. 147: 158-172. PubMed

Nagel, C. H., Albrecht, N., et al. (2011). Herpes Simplex Virus Immediate-Early Protein ICP0 Is Targeted by SIAH-1 for Proteasomal Degradation. Journal of virology. 85: 7644. PubMed

Studencka, M., Konzer, A., et al. (2011). Novel roles of C. elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression. Molecular and Cellular Biology.PubMed

Muehlen, S., Ruchaud-Sparagano, M. H., et al. (2011). Proteasome-independent Degradation of Canonical NFŒ?B Complex Components by the NleC Protein of Pathogenic Escherichia coli. Journal of Biological Chemistry. 286: 5100. PubMed

Galan, J. A., Paris, L. L., et al. (2011). Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap. Journal of the American Society for Mass Spectrometry. 1-10. PubMed

Chamousset, D., De Wever, V., et al. (2010). RRP1B Targets PP1 to Mammalian Cell Nucleoli and is Associated with Pre-60S Ribosomal Subunits. Mol Biol Cell. PubMed

Kovacs, E. M., Verma, S., et al. (2011). N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nature Cell Biology. 13: 934-943. PubMed

Boysen, K. E. and Matuschewski, K. (2011). Arrested oocyst maturation in Plasmodium parasites lacking type II NADH: ubiquinone dehydrogenase. Journal of Biological Chemistry. 286: 32661-32671. PubMed

Mortusewicz, O., Fouquerel, E., et al. (2011). PARG is recruited to DNA damage sites through poly (ADP-ribose)-and PCNA-dependent mechanisms. Nucleic acids research. 39: 5045. PubMed

Graewe, S., Rankin, K. E., et al. (2011). Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathogens. 7: e1002224. PubMed

Yang, X. D., Huang, S., et al. (2011). Distinct and mutually inhibitory binding by two divergent Œ?-catenins coordinates TCF levels and activity in C. elegans. Development. 138: 4255-4265. PubMed

Pollithy, A., Romer, T., et al. (2011). Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense. Applied and environmental microbiology. 77: 6165-6171. PubMed

Kozubowski, L., Thompson, J. W., et al. (2011). Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics. PLoS One. 6: e25280. PubMed

Garcia-Gomez, J. J., Lebaron, S., et al. (2011). Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae. Molecular and Cellular Biology. 31: 4156-4164. PubMed

Van Damme, D., Gadeyne, A., et al. (2011). Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proceedings of the National Academy of Sciences. 108: 615. PubMed

Qvist, P., Huertas, P., et al. (2011). CtIP Mutations Cause Seckel and Jawad Syndromes. PLoS Genetics. 7: e1002310. PubMed

Labella, S., Woglar, A., et al. (2011). Polo Kinases Establish Links between Meiotic Chromosomes and Cytoskeletal Forces Essential for Homolog Pairing. Developmental Cell. PubMed

Harterink, M., Port, F., et al. (2011). A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biology. 13: 914-923. PubMed

Konopacki, F. A., Jaafari, N., et al. (2011). Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis. Proceedings of the National Academy of Sciences.PubMed

Chuhma, N., Tanaka, K. F., et al. (2011). Functional connectome of the striatal medium spiny neuron. The Journal of Neuroscience. 31: 1183-1192. PubMed

Jackson, B. R., Boyne, J. R., et al. (2011). An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export. PLoS Pathogens. 7: e1002138. PubMed

Tags: anti-GFP, camelid antibodies, Camelid Antibody, Chromotek, Co-IP, GFP-Trap, GFPTrap, immunoprecipitation, pulldown, RFPTrap, VHH

Year-end message from Allele Biotech

The year 2011 has been an exciting and eventful year for many people. Throughout the year, we have been working diligently to bring the best research methods in many areas to our fellow researchers through innovation and entrepreneurship. Thanks in part to the government’s stimulus and grant support in 2011, we established several new product lines, including the Stealth iPS induction mRNA templates and reagents, a great new photoconvertible fluorescent protein in mClavGR2 (through collaboration with academic colleagues), and a highly efficient lentivirus-based shRNA packaging service as a result of an NCI SBIR contract.

As you all must have noticed by now, in July we redesigned our website to present our products in an easier, more user friendly manner, while adding a convenient online purchasing system. We have received a lot of positive feedback from customers telling us how “cool” the new site is, and how easy it is to use and redeem promotions. Towards the end of the year, our dedicated marketing and sales teams reinstated our biweekly email newsletters (to receive our messages on new discoveries and technologies, or be the first to use our promotions, sign up online under “Newsletter”).

All of these efforts would have been meaningless without our customers, who ultimately gave us the opportunity to be in the business we love and are trained to do. By selecting our products, sending us feedback, and “retweeting” or “reposting” our messages, you have been tremendously valuable to every one of us here at Allele. We thank you from the bottom of our hearts. In return, we will continue to invest and do our very best to provide new tools for advancing your research. Watch for our brand new monomeric fluorescent protein that can be nearly 10 times brighter than EGFP; a more powerful iPSC generation method that could potentially reprogram in just a few days, and much much more in 2012!

Tags: 2012, EGFP, iPS, iPSC, mRNA templates, NCI SBIR, new fluorescent proteins, reposting, retweeting

Top 10 List of Most Viewed AlleleBlogs in 2011

The ballot is in—among the “usual suspect” hot topics, iPS takes the top honor and most entries; Camelid antibodies, although not really presented as a typical AlleleBlog in 2011, made it to the top 3. shRNA cloning and RNAi screening are still on a lot of people’s minds, so it seems.

Method: total visits to each blog since our new webpage was launched in July was counted.

1) Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming
http://blog.allelebiotech.com/2011/10/fusion-of-the-transcription-domain-to-ips-factors-radically-enhances-reprogramming/

2) Methods of iPSC Generation Update
http://blog.allelebiotech.com/2011/08/methods-of-ipsc-generation-update/

3) About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011
http://blog.allelebiotech.com/2011/09/about-50-papers-cited-the-use-of-gfp-trap-camelid-antibody-so-far-in-2011/

4) Big Potential in Using Protozoans for Producing Mammalian Proteins
http://blog.allelebiotech.com/2011/09/big-potential-in-using-protozoans-for-producing-mammalian-proteins/

5) How do you make shRNA-expressing viruses for function screening?
http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

6) Creating ground-state human iPSCs
http://blog.allelebiotech.com/2011/10/creating-ground-state-human-ipsc/

7) Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
http://blog.allelebiotech.com/2011/03/recombinase-mediated-cassette-exchange-rmce-and-integrase-swappable-in-vivo-targeting-element-insite/

Development of Cell Lines from iPSCs for Bioassays
http://blog.allelebiotech.com/2011/11/development-of-cell-lines-from-ipscs-for-bioassays/

9) Choosing siRNA, shRNA, and miRNA for Gene Silencing
blog.allelebiotech.com/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/

10) Allele Biotech’s Box Swap Program
http://blog.allelebiotech.com/2009/07/allele-biotechs-box-swap-program/

Have a successful 2012!

Tags: camelid antibodies, environment friendly research, GFP-Trap, iPS, mRNA iPS, RiPSC, RNAi screening, shRNA lentivirus packaging

How do you make shRNA-expressing viruses for function screening?

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

New Products of the week: 100x 15mm EcoCulture Vented Dishes for better stem cell attachment and less plastic waste to the environment, APB-CS-114TC.

Promotion of the week: Buy 1 Stealth Express IPS Induction PCR Template Set, get 1 SurfaceBind RNA Purification Kit free. Use code FreePureRNA.

Tags: lentivirus packaging, lentiviurs shRNA, RNAi, RNAi screening, shRNA, shRNA cloning, shRNA lentivirus packaging, shRNA library

Development of Cell Lines from iPSCs for Bioassays

The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. Using immortalized cell lines for drug screening assays has its limitations, such as questionable relevance; and the use of primary cells is often hindered by supply difficulties. Thanks to pioneering work by the Yamanaka, Thompson, and other groups, the feasibility of creating iPSCs has generated an opportunity to provide cell lines with stem cell properties in a virtually unlimited supply [1, 2]. These cells can be derived into different cell types for specific assays, even with patient- or genotype-specific background. Technologies are being developed to produce re-differentiated cells of a number of lineages.

Take cardiomyocytes as an example. There are a number of conventional methods for inducing stem cells into cardiomyocytes: through embryoid body (EB) formation, co-culturing with visceral endoderm-like cell line (END-2), and monolayer caridomyocyte differentiation with defined growth medium and protein factors [3]. A recent publication showed that using appropriate concentrations of BMP4 and activin-A in BSA-containing medium cardiomyocytes might be achieved from iPSCs or ESCs in about 6 days [4].

Transdifferentiation, or direct reprogramming, by introducing a group of 3 cardiomyocyte-specific factors, investigators could directly program cardiac or dermal fibroblasts into cardiomyocyte-like cells [5]. Although much refinement and characterization of these directly reprogrammed cardiomyocyte-like cells, termed iCMs, will be needed before the process can become widely used, this work raised the possibility of quicker and perhaps more efficient ways of generating cells for assays. Similar transdifferentiation has resulted in induced neuron (iN) cells, also by introducing 3 tissue-specific transcription factors [6]. Therefore, it seems that by using defined combinations of tissue-specific transcription factors it is possible to generate cells of different tissue types. It is also possible that by using different, developmental stage-specific transcription activator sets, transdifferentiation can be conducted in a stepwise way and make sure cells at each step is pure. This strategy may be particularly attractive if its efficiency can be improved by the techniques developed for iPSC creation. After all, reprogramming to pluripotency and transdifferentiation to different tissue types must share certain mechanistic steps in their respective processes.

In addition, it has been reported that by briefly overexpressing the Yamanaka iPS factors and controlling growth conditions, mouse fibroblasts could be transdifferentiated up to 40% in 18 days without reversing back to pluripotency [7]. It would be interesting to see if by transient expression of iPS factors via mRNA then switching to cardiomyocyte-specific transcription factors, we can increase the efficiency for direct reprogramming. Use of chromatin-modifying chemicals that were already shown to directly reverse and alter cell fates might also be used to assist direct reprogramming. We believe that a systematic approach for studying these reprogramming aspects should benefit the iPS fields.

1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Vidarsson, H., J. Hyllner, and P. Sartipy, Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev, 2010. 6(1): p. 108-20.
4. Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011.
5. Ieda, M., et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010. 142(3): p. 375-86.
6. Pang, Z.P., et al., Induction of human neuronal cells by defined transcription factors. Nature, 2011. 476(7359): p. 220-3.
7. Efe, J.A., et al., Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 2011. 13(3): p. 215-22.

New Products of the week: T7 RNA Polymerase, high quality for demanding in vitro transcription requirements.

Promotion of the week: GFP-Trap, buy 2 of any package and get 1 of equal or less value free. Use code FreeTrap, follow deals quickly on Facebook.

Tags: cardiomyocyte, didifferentiation, direct reprogramming, Gata4, iCM, iN, iPSC, Mef2c, redifferentiation, Tbx5, transdifferentiation