Marker Gene Monthly Newsletter - Volume 11, Number 10 - October, 2011

Multiparameter FACS Analysis

Flow cytometry has become one of the most important tools in immunology. In flow cytometry, cells are first labeled with a fluorescent protein or dye, or with a fluorescently labeled antibody and then brought up into a cell suspension. Droplets of this suspension are passed through a lens system in which a beam of light is used to activate the fluorescent dye. The cell suspension is adjusted so that each droplet that passes through the detector contains only one cell, and therefore the labeling of the cells with different dyes or antibodies can be quantitatively measured. This method can therefore be used to monitor cell metabolism, the transfection of different genes, or to identify different cell types. Current methods utilize laser excitation to monitor specific wavelengths and therefore specific dye labeling. But the technique is often limited by spectral overlap of the dyes and the number of different colors (wavelengths) that can be simultaneously measured.

Recent work from the laboratory of Scott Tanner at the University of Toronto have sought to improve the multiplexing limitation of current flow cytometry by utilizing antibodies that are labeled with chelators that bind to specific heavy-metal isotopes. The isotopes are then detected and quantitated using a mass spectrometry technique (ion-coupled plasma mass spectrometer, ICP-MS). In this way up to 35 different markers can be simultaneously measured and with greater accuracy than current techniques. In addition, there is no need to correct for background fluorescence sometimes found in cells using fluorescence based techniques. Although still in developmental stages, the technology is already commercially available as the CyTOF instrument from DVS Sciences. For more information about these techniques, please see the references below or visit our website

• Ornatsky O, Bandura D, Baranov V, Nitz M, Winnik MA; Tanner S (2010). "Highly multiparametric analysis by mass cytometry". Journal of Immunological Methods 361 (1–2): 1–20.
• Bendall SC, Simonds EF, Qiu P, Amir ED, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe’er D, Tanner SD, Nolan GP (2011) "Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science 6 May 2011: 687-696.
• Krutzik PO, Nolan GP, (2006) "Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling." Nat. Methods 3: 361.
• Chattopadhyay PK, Price DA, Harper TF, Betts MR, Yu J, Gostick E, Perfetto SP, Goepfert P, Koup RA, De Rosa SC, Bruchez MP, Roederer M, (2006) "Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry." Nature Med. 12(8): 972-977.

RNA Sequences that Switch on the Fluorescence of Small Molecules

Protein synthesis is controlled in the cell by mRNA expression and translation. But much of the human genome codes for sequences that are not utilized directly for protein synthesis. These so-called non-coding regions, however, have been found to have significant importance in gene regulation, epigenetic analysis and for tracking cellular processes and differentiation. Recently Dr. Samie R. Jaffrey, and co-workers at the Department of Pharmacology, Weill Medical College, Cornell University have developed an elegant method for monitoring RNA dynamics in live cells by utilizing a synthetic version of the nascent fluorophore derived from GFP (4-hydroxybenzene imidazolinone (HBI)). The HBI molecule itself is non-fluorescent, but when held into a specific conformation by binding to an RNA sequence, fluorescence becomes the major pathway available to dissipate the energy of the excited state fluorophore. In addition, through screening they were able to identify specific RNA aptamer sequences that were best suited to bind to the HBI or the more efficient dimethoxy (DMHBI) or difluoro (DFHBI) synthetic analogs. Several aptamers were identified that exhibited markedly different spectral properties, including emitting a blue, greenish-yellow, yellow and and even red fluorescence. By cloning the aptamer sequences as fusions with existing RNA sequences, they were able to track the movements of individual RNAs, such as the 5S RNA in HEK293 T cells, intracellularly. Additional small molecule fluorophores based upon the RFP fluorophore, for example, are under development. The dyes have been found to exhibit good resistance to photobleaching and the enhancement of fluorescence upon binding is quick. The combined application of these unique small molecule dyes and RNA aptamers to trace RNA trafficking as well as potentially monitoring RNA-RNA and RNA-protein interactions is significant. To find out more about these methods, please see references below or visit our website.

• Paige JS, Wu KY, Jaffrey SR (2011) "RNA mimics of green fluorescent protein." Science 333:642-646.
• Mattick JS, Clark MB (2011) "RNA lights up." Nature Biotechnology 29: 883–884.
• Park HY, Buxbaum AR, Singer RH. (2010) "Single mRNA tracking in live cells." Methods Enzymol. 472:387-406.

NF-kB and Senescence Analysis in Cancer

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor that is involved in the activation of a number of genes in response to cell stimuli including antigen presentation, stress, viral expression, oxidative processes, free radicals, UV radiation and cytokine binding. It has been long believed that the activation of NF-κB causes the upregulation of a variety of genes that are correlated with tumorigenesis including proliferation, invasion and metastasis of tumors.  Moreover, increased levels of NF-κB have been found in a variety of cancers such as prostate, breast, ovarian tumors as well as leukemeas, lymphomas and myelomas. Thus NF-κB has become an attractive target for cancer therapy and indeed drugs have been developed to inhibit its activity. However, recent work from the laboratories of Scott Lowe and collegues at the Sloan-Kettering Cancer Center, New York has found the other “face” of NF-κB; that is, NF-κB is required to promote cancer cells undergoing senescence.  Indeed, Clements Schmitt and colleagues at the Charite University Hospital in Berlin discovered that active NF-κB signaling is required for sensitivity to chemotherapeutic drugs in patients.
Measurement of the NF-κB transcription factor is typically accomplished using a dual-luciferase assay, in which one luciferase, from Firefly for example, is linked to an NF-κB recognition site and another luciferase (e.g. Renilla) under constitutive promoter is used as a control to normalize transfection efficiency. Activation of expression is then calculated as the relative Firefly luciferase activity normalized with respect to the activity of transfection of the control Renilla luciferase. In addition, senescence assays can be performed in the cells or tissue samples by measuring the amount of senescence associated b-Gal activity (SA-bGal) that is increased when the cells enter into senescence. The combined assays may be important in monitoring the effectiveness of transcription factor targeted therapies using high-throughput analysis techniques. For more information about these techniques, please see the references below or visit our website

• Chien Y, Scuoppo C, Wang X, Fang X, Balgley B, Bolden JE, Premsrirut P, Luo W, Chicas A, Lee CS, Kogan SC, Lowe SW (2011) "Control of the senescence-associated secretory phenotype by NF-k B promotes senescence and enhances chemosensitivity." Genes Dev. 25: 2125-2136.
• Jing H, Kase J, Dorr JR, Milanovic M, Lenze D, Grau M, Beuster G, Ji S, Reinmann M, Lenz P, Hummel M, Dorken B, Lenz G, Scheidereit C, Schmitt CA, Lee S (2011) “Opposing roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investigation.” Genes Dev. 25:2137-2146.
• Escárcega RO, Fuentes-Alexandro S, García-Carrasco M, Gatica A, Zamora A (2007). "The transcription factor nuclear factor-κB and cancer". Clinical Oncology (Royal College of Radiologists (Great Britain)) 19 (2): 154–61.
• Galardi S, Mercatelli N, Farace MG, Ciafre` SA (2011) "NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells." Nucleic Acids Research 39(9): 3892-3902.

GUS Assay for Recombinant Plant Analysis

The ß-glucuronidase (GUS) gene isolated from E. coli (EC 3.2.1.31) has been well documented to provide desirable characteristics as a marker gene in transformed plants.  It has been among the most widely utilized marker genes for plant biotechnology, and a great number of analysis techniques have been developed for its use in a wide variety of plant systems. Although there are some reports of endogenous inhibitors as well as slight nascient glucuronidase activity present in some plant tissues, it still remains one of the easiest and most sensitive marker gene systems available for plant use.

Our new ß-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) provides all the reagents, buffers, and a detailed protocol for easy quantitative measure of GUS enzyme activity in transformed plants or plant cells, through use of the fluorogenic substrate 4-methylumbelliferyl b-D-glucuronic acid (M0240).   Plants or other cell types are extracted with GUS extraction buffer containing phosphate-EDTA, pH 7.0 and detergents. The extracted b-glucuronidase hydrolyzes the 4-MUG to the fluorescent compound 4-MU (pKa 8.2) and glucuronic acid. The reaction is stopped with sodium carbonate buffer because 4-MU exhibits maximal fluorescence at pH values above its pKa. 4-MU can be excited at 365nm with emission maximum at 455nm. For more information about these techniques, please see the references below or visit our website

• Horvath BM, Magyar Z, Zhang Y, Hamburger A, Bako L, Visser RGF, Bachem CWB, Bogre L. (2006) “EBP1 regulates organ size through cell growth and proliferation in plants.” EMBO J. 25: 4909-4920
• Jefferson RA, Burgess SM, Hirsh D (1986) "beta-Glucuronidase from Escherichia coli as a gene-fusion marker." Proc. Natl. Acad. Sci. USA. 86: 8447-8451.
• Jefferson RA, Kavanagh TA, Bevan MW (1987) "GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants." EMBO J. 9(13): 63901-3907.
• Fior S, Gerola PD (2009) "Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS." Plant Methods 5:19.
• Kosugi, S, Ohashi Y, Nakajima K, Arai Y (1990) "An impoved assay for b-glucuronidase in transformed cells: methoanol almost completely supresses putative endogenous b-glucuronidase activity." Plant Sci. 70: 130-140.
• Naleway, J.J. Chap. 4. in “GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression”. Gallagher, S.R., ed. Acad/ Press, NY, (1992).
  

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