Marker Gene Monthly Newsletter
September, 2008
Volume 8, Number 9
© Copyright MGT, Inc., 2008. Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA. All rights reserved. For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com. Please see below for subscription information and updates. This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Doxycycline Inducible Somatic Cell Reprogramming

The ability to reprogram mammalian differentiated cells into pluripotent embryonic stem (ES) cells is an exciting new method that has recently been accomplished in human fibroblasts by introducing a set of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, using retroviral transfer under ES cell culture conditions.  But the levels of expression of these genes and genetic heterogeneity in the reprogrammed cells typically leads to low frequencies of induced pluripotent stem (iPS) cells. Recent work from the laboratory of Rudolf Jaenisch at the Whitehead Institute of Biomedical Research at MIT designed an elegant system by which these four transcription factors were placed under the control of a drug-inducible promoter (dox) in lentiviral vectors. After viral transduction, doxycycline was added to the culture medium to activate the transgenes and initiate the reprogramming process. Using this system they were able to ascertain the kinetics necessary for reprogramming by using a Nanog-GFP reporter system. By exposure to doxycycline for between 5-22 days, they determined that the minimum length of reprogramming was about 9 days, and after 16 days, no more iPS cells were obtained. Interestingly, by fine tuning these techniques, they were able to increase the efficiency of reprogramming by 25-50X that for direct infection techniques with drug selection. They were able to demonstrate these techniques in a variety of human cell lines derived from the kidney, adrenal gland, muscle, neural, intestinal epithelium and other cell lines. In addition, the added level of control in this method will allow studies into the molecular events that lead to epigenetic reprogramming. For more information about these systems, please see the references below or visit our website.

• Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, Staerk J, Markoulaki S, Jaenisch R (2008) "A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types." Nature Biotechnology 26: 916 - 924.
• Park, IH, Zhao R , West JA, Yabuuchi A, Huo H , Ince TA, Lerou PH , Lensch MW, Daley GQ, "Reprogramming of human somatic cells to pluripotency with defined factors." Nature 451: 141–146.
• Lowry, WE, Richter L, Yachechko L, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K, "Generation of human induced pluripotent stem cells from dermal fibroblasts." Proc. Natl. Acad. Sci. USA 105: 2883–2888.
• Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S."Induction of pluripotent stem cells from adult human fibroblasts by defined factors." Cell 131: 861–872.

Holographic Activation of Caged Neurotransmitters

Probing the function of synaptic glutamate receptors is important for understanding the basic mechanisms of synaptic transmission in neural networks. Glutamate receptors are unevenly distributed on the plasma membrane of neurons and methods of analyses of receptor function can be greatly aided by the ability to map these glutamate receptors. Recently, work from the laboratory of Dr. Valentina Emiliani at the University of Paris and co-workers developed a nematic liquid-crystal spatial light modulator that uses holographic patterns of illumination to excite specific areas of neurons. Using this holographic-patterned light excitation, they were able to activate caged-glutamate compounds in specific sections of individual neurons. The ability to release caged compounds using light excitation has been known and the resulting activated receptors can be visualized with IgG-conjugated Alexa594 or other fluorescent dyes (Fluo-4). Using the new caged compound MNI-glutamate (4-methoxy-7-nitroindolino-glutamate) they were able to show spacial activation of AMPA receptors on neurons. A similar two-photon IR illumination system was also recently developed by Matsuzaki, et al. (2001). But such holographic excitation and activation systems have the potential to allow photostimulation in specific patterns or in various planes of cells or tissue, and also can be used in other techniques such as FRAP or with photoactivatable fluorescent proteins. For more information about these systems, please see the references below or visit our website.

• Lutz C, Otis TS, DeSars V, Charpak S, DiGregorio DA, Emiliani V, (2008) "Holographic photolysis of caged neurotransmitters." Nature Methods 5(9): 821-827.
• Morrison, J., Wan, P., Corrie, J. E. & Papageorgiou, G. "Mechanisms of photorelease of carboxylic acids from 1-acyl-7-nitroindolines in solutions of varying water content." Photochem Photobiol Sci 1, 960-9 (2002).
• Canepari, M., Nelson, L., Papageorgiou, G., Corrie, J. E. & Ogden, D. Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinylamino acids as novel, fast caged neurotransmitters. J Neurosci Methods 112, 29-42 (2001).
• Matsuzaki, Masanori; Ellis-Davies, Graham C. R.; Nemoto, Tomomi; Miyashita, Yasushi; Iino, Masamitsu; Kasai, Haruo (2001) "Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons." Nature Neuroscience 4(11): 1086-1092.

Reversibility in Cell Quiescence

Cellular quiescence is distinct from senescence or terminal differentiation in cell growth in that in quiescence, the cells retain the ability to restore normal proliferation and growth. Cells entering each of these stages stop their cell division and cell cycle events, usually by expression and accumulation of inhibitory compounds, such as cyclin-dependent kinase inhibitors. Recent work from the laboratory of Dr. James Roberts at the Fred Hutchison Cancer Research Center at the University of Washington has pointed to the transcriptioinal respressor HES1 that codes for a basic helix-loop-helix protein as required for quiescence to be reversible. By cloning in the HES1 factor into early passage lung fibroblasts and also cloning in a vector containing the CDK inhibitor p21 they were able to monitor the effect of the Hes1 protein. Previous experiments had shown that sustained expression of p21 caused cell cycle arrest, even for brief periods (4 days), making the cells enter irreversible senescence. Senescence was measured using the senescence-associated b-galactosidase assay (SA-b-Gal, M1389). But the Hes1 expression was able to restore cell growth and cause them to be resistant to terminal differentiation and cell cycle arrest (in concert with other procedures). In the same manner, inactivation of Hes1 caused non-dividing cells to spontaneously enter into an irreversible senescent state. Because many types of cells including stem cells, as well as several types of cancer cells including ovarian carcinomas, breast cancers, non-small cell lung cancers and others have been shown to have increased Hes1 levels, the implications for use in maintaining stem cell lineages as well as in promoting or inhibiting tumorigenisis are significant. For more information about these systems, please see the references below or visit our website.

• Sang L, Coller HA, Roberts JM, (2008) " Control of the reversibility of cellular quiescence by the transcriptional repressor HES1." Science 321(5892):1095-100.
• Yu X, Alder JK, Chun JH, Friedman AD, Heimfeld S, Cheng L, Civin CI, (2006) "HES1 inhibits cycling of hematopoietic progenitor cells via DNA binding." Stem Cells 24(4):876-88.
• Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowsk, D, Sykes M, Scadden DT (2000). "Hematopoietic Stem Cell Quiescence Maintained by p21cip1/waf1." Science 287: 1804-1808.
• Dimri GP, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubeli I, Pereira-Smith O, Peacocke M, Campisi J (1995) "A biomarker that identifies senescent human cells in culture and in aging skin in vivo." Proc. Natl. Acad. Sci. USA 92: 9363-9367.

Improved FRET Quencher for FRET Substrates

FRET assays incorporate a fluorophore-quencher pair wherein the quencher substantially absorbs the excitation wavelengths of light that are required for fluorophore excitation, as long as the distance between the pair is small. This distance, the so-called Forster radius, is usually about 10 to 100 angstoms in length. But these distances make FRET a very attractive technique for use in designing quenched protease substrates for endopeptidase activities. By attaching the fluorophore to one end of the peptide, and the quencer to the other side, fluorescence can be essentially quenched until the peptidase activity cleaves the specific protease site, and releases the two fragments. Since they are no longer in close proximity, the fluorescence levels will increase, proportional to enzyme activity. Common FRET fluoropohore-quencher pairs have included EDANS/DABCYL (M0084) (M1051) and amp/Dnp. Recently a new quencher QXL 520 has been developed that has an absorption spectrum which more closely matches that of fluorescein, and this induces more specific and efficient quenching over almost the entire spectrum of the dye and results in a decrease in background signal for such FRET substrates. Use of this new quencher in the design of improved substrates for various enzyme activities presents an attractive new area for research. For more information about these systems, please see the references below or visit our website.

• Förster, T. (1948) Intermolecular Energy Migration and Fluorescence Ann. Phys 2:55-75.
• Po CL, Rakmanova V, (2008) "Sensitive FRET Assays Aid Drug Discovery." Biophotonics Intl. 15(8): 34-35.
• Pennington MW, Thornberry NA (1994) "Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer." Pept Res. 7: 72-76 .
• Wang GT, Krafft GA(1992) "Automated synthesis of fluorogenic protease substrates: design of probes for alzheimers disease-associated proteases." Bioorg Med Chem Lett. 2: 1665.
• Maggiora LL, Smith CW, Zhang ZY (1992) "A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis." J Med. Chem. 35: 3727-3730.

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