Marker Gene Monthly Newsletter   

December, 2006

Volume 6, Number 12

© Copyright MGT, Inc., 2007.  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.

PET Analysis of Marker Genes.

The use of marker genes such as luciferase, b-galactosidase and green fluorescent protein have opened many new areas in cell biology research. Recent work from the laboratory of Dr. Harvey Herschman and collaborators at the Crump Institute for Molecular Biology, UCLA School of Medicine have reported development of new marker gene systems that can be used to image gene expression in living animals. They employ marker genes whose protein products can act upon positron labeled substrates, and then image the marker gene position and concentration by trapping of the positron labeled reporter probes in the transgenic tissues.  The analysis is performed using very sensitive positron emission tomography (PET) techniqes.   These systems are based upon the well known  2-deoxy-2-[18F]-fluoro-D-deoxyglucose (FDG) methods of measuring metabolism non-invasively, in living subjects using PET.  Hexokinase converts FDG to a phosphorylated product that is trapped inside cells. FDG is transported into, phosphorylated and retained in tissues—where its accumulation can be measured tomographically—in proportion to the glycolytic rate. Using PET, properties such as receptor density and metabolic rate can be repeatedly and noninvasively measured in living animals.

Two non-invasive PET marker gene imaging reporter systems have been developed.  The first is an enzyme-based system that utilizes the Herpes Simplex Virus thmidine kinase gene (HSV1-tk) as marker gene and positron labeled substrates as the reporter probes.   The cloned HSV-tk enzyme acts to phosphorylate these various nucleoside analogs that, upon phosphorylation, become “trapped” intracellularly due to their increased charge.  Positron-emitting analogs of known HSV1-tk substrates were synthesized, and modified HSV1-TK enzymes developed that permitted in vivo monitoring of HSV1-tk levels in transfected tissues by positron emission tomography (PET) through measurement of the enzymatically phosphorylated products. Imaging HSV1-tk expression with the uracil nucleoside derivative 5-[¹²⁴I]iodo-29-fluoro-29-deoxy-1-b-D-arabino-5-iodouracil ([¹²⁴I]FIAU) and with the acycloguanosine derivatives 8-[¹⁸F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine([¹⁸F]GCV), 8-[¹⁸F]fluoro-9-[4-hydroxy-3-(hydroxymethyl)-1-butyl] guanine ([¹⁸F]PCV) as well as 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl) butyl]guanine ([¹⁸F]FHBG) were performed.  These derivatives could be prepared chemically using protected nucleoside intermediates.
 
The second system is a receptor-based method that uses the Dopamine D2 Receptor as the marker gene and a positron labeled ligand as the reporter probe. Specifically, 3-(29-[¹⁸F]fluoroethyl)Spiperone (FESP) is a ligand that binds to the dopamine D2 receptor (D2R). When FESP is injected intravenously, it accumulates in tissues expressing D2R.  Images are then collected in the tomograph, and D2R-rich tissues can be identified, D2R levels measured and the levels of secondary transcribed genes inferred. These PET techniques have been used to detect as low as 2 nM concentrations of D2R in the neural tissues of living animals. For more information about these new techniques, please visit our website or see the references below.

• I.Y. Chen, J.C. Wu, J.J. Min, G. Sundaresan, X. Lewis, Q. Liang, H.R. Herschman, S.S. Gambhir, (2004) “Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery” Circulation 109(11): 1415-20.
• M. Iyer, J. Barrio, M. Namavari, E. Bauer, N. Satyamurthy, K. Nguyen, T. Toyokuni, M.E. Phelps, H.R. Herschman, S.S. Gambhir, (2001) “8-[18F]Fluoropenciclovir: An Improved Reporter Probe for Imaging HSV1-tk Reporter Gene Expression In Vivo Using PET.” Journal of Nuclear Medicine. 42(1): 96-105.
• Q. Liang, N. Satyamurthy, J.R. Barrio, T. Toyokuni, M.E. Phelps, S.S. Gambhir, H.R. Herschman. (2001) “Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction.” Gene Therapy 8(19): 1490-1498.
• S.S. Yaghoubi, J.R. Barrio, M. Dahlbom, M. Iyer, M. Namavari, N. Satyamurthy, R. Goldman, H.R. Herschman, M.E. Phelps, S.S. Gambhir (2001) “Human Pharmacokinetic and Dosimetry Studies of [18F]FHBG: A Reporter Probe for Imaging Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression” Journal of Nuclear Medicine 42(8): 1225-1234.
• S.S. Yaghoubi, L. Wu, Q. Liang, T. Toyokuni, J.R. Barrio, M. Namavari, N. Satyamurthy, M.E. Phelps, H. Herschman, S.S. Gambhir. 2001; “Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.” Gene Therapy 8(14): 1072-1080.
• X. Sun, A.J. Annala, S. Yaghoubi, J.R. Barrio, K. Nguyen, T. Toyokuni, N. Satyamurthy, M. Namavari, M.E. Phelps, H.R. Herschman, S.S. Gambhir, (2001) “Quantitative imaging of gene induction in living animals.” Gene Therapy 8(20): 1572-1579.
• D. MacLaren, T. Toyokuni, S. Cherry, J. Barrio, M. E. Phelps, H. R. Herschman, S.S. Gambhir (2000) “PET Imaging of Transgene Expression” Biological Psychiatry. 48(5): 337-348.
• H.R. Herschman, D.C MacLaren, M. Iyer, M. Namavari, K. Bobinski, L.A. Green, L. Wu, A.J. Berk, T. Toyokuni, J.R. Barrio, S.R. Cherry, M.E. Phelps, E.P. Sandgren, S.S. Gambhir. “Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography.” (2000) Journal of Neuroscience Research. 59(6): 699-705.
• M. Namavari, J.R. Barrio, T. Toyokuni, S.S. Gambhir, S.R. Cherry, H.R. Herschman, M.E. Phelps, N. Satyamurthy, (2000) “Synthesis of 8-[18F] Fluoroguanine Derivatives: In-vivo Probes Imaging Gene Expression with PET” Nuclear Medicine and Biology 27(2): 157-162.
• S.S. Gambhir, E. Bauer, M. Black, Q. Liang, M.S. Kokoris, J. Barrio, M. Iyer, M. Namavari, M.E. Phelps, H.R. Herschman (2000) “A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography” Proc Natl Acad Sci. 97(6): 2785-2790.
• S.S. Gambhir, H.R. Herschman, S.R. Cherry, J.R. Barrio, N. Satyamurthy, T. Toyokuni, M.E. Phelps, S.M. Larson, J. Balatoni, R. Finn, J. Tjuvajev, R. Blasberg (2000) “Imaging Transgene Expression with Radionuclide Imaging Technologies” Neoplasia. 2(1-2): 118-138.
• Y. Yu, A.J. Annala, J.R. Barrio, T. Toyokuni, N. Satyamurthy, M. Namavari, S.R. Cherry, M.E. Phelps, H.R. Herschman, S.S. Gambhir. (2000) “Quantification of target gene expression by imaging reporter gene expression in living animals” Nature Medicine 6(8): 933 – 937. · 
• D.C. MacLaren, S.S. Gambhir, N. Satyamurthy, J.R. Barrio, S. Sharfstein, T. Toyokuni, L. Wu, A.J. Berk, S.R. Cherry, M.E. Phelps, H. Herschman, (1999) “Repetitive, non-invasive imaging of the dopamine D-2 receptor as a reporter gene in living animals” Gene Therapy 6(5): 785-791.
  

Photobleaching and Blinking of Fluorophores.

Fluorophores have been known to exhibit spontaneous fluctuations in emission, that are sometimes difficult to distinguish from actual enzymatic turnover when used in enzyme assay systems.  These fluctuations are termed flickering (for unresolved bands) or blinking (where discrete dark states exist for up to a number of milliseconds to seconds).  There appear to be several causes of these emission oscillations. Triplet-state formation can be a contributing factor that can operate over a wide range of time scales.  But polarization effects are also important at the single-molecule level, where slow rotations of the fluorophore (compared with the acquisition time) can give rise to changes in the efficiency of excitation and emission values.  A more significant problem occurs where the fluorophore can exist as several photo-induced isomer forms.  It is well known that excitation of highly-conjugated organic dyes can cause photoisomerization because of the large number of pi-electron orbitals in the system.  If the absorption spectra of the isomers differ, then excitation at a single wavelength can influence the population towards one isomer (the so-called photochromic effect).  One isomer might then become trapped in a dark state for an amount of time, especially if it does not efficiently absorb at the excitation wavelength, and the ground-state barrier for the isomer transition is high.  Slow blinking is particularly problematic when it overlaps with biologically relevant time scales.  If the recovery time is on the same time scale as the enzyme kinetics, for example, the blinking can be difficult to distinguish from the actual enzyme turnover. 

 

It has been proposed that blinking need not be associated with intrinsic properties of the fluorophore but can depend upon the local physical environment.  Protonation of a fluorophore can cause several isomeric structures to exist.  Increases in excitation intensity can then also increase the blinking rate, because photon absorption can drive this protonation cycle. For example, GFP blinks on the millisecond time scale, which is in line with known protonation studies. In fact, GFP, will emit fluorescence only in the protonated state.  When two GFP molecules dimerize, the low fluorescence unprotonated state of one can even become more favorable and blinking can become more prevalent on a longer time scale.  This phenomenon can lead to difficulties in counting the number of GFP subunits in a single molecular complex from simple intensity measurements.  The cyanine dye, Cy5, also has shown general blinking properties.  Cy5 blinking is affected by the presence of oxygen or oxygen scavengers, indicating that triplet-states are the possible source of  blinking.  An enzymatic oxygen-scavenging system can be used to eliminate CY5 blinking, and can also dramatically reduce photobleaching and improve the signal linearity at high excitation rates.  Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid) in combination with an enzymatic oxygen-scavenging system (a mixture of glucose oxidase and catalase) can significantly extend the applications of such fluorescent dye labeling techniques.  For more information about these reagents for preventing blinking of your fluorescently labeled samples, see the references below or visit our website.

• Bock G., Hilchenbach M., Schauenstein K., Wick, G. “Photometric analysis of antifading reagents with laser and conventional illumination sources.” J. Histochem. & Cytochem. 33(7): 699-705, 1985.
• C.R.Bagshaw C.R., Cherny D., (2006) “Blinking florophores: what do they tell us about protein dynamics?” Biochem. Soc. Trans. 34(5): 979-984.
  
• Johnson G.D. and De C Nogueira Araujo G.M. A simple method for reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods. 43:349, 1981.
  
• Rasnik, I., McKinney S.A., Ha T., (2006) “Nonblinking and long-lasting single-molecule fluorescence imaging.” Nature Methods 3: 891 – 893. 
  
• Johnson G.D., Davidson R.S., McNamee K.C., Russell G., Goodwin D., and Holborow E.J. Fading of Immunofluorescence during microscopy: a study of the phenomenon and its remedy. J. Immunol. Methods. 55:231, 1982.
•  Krenik K.D., Kephart G.M., Offord K.P.,Dunnette S.L. and Gleich, G.J. Comparison of antifading reagents used in immunofluorescence. J. Immunol. Methods. 117:91-7, 1989.
• Longin A., Souchier C., French M., Bryon PA. Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J. Histochem. & Cytochem. 41(12):1833-40, 1993 Dec.

EDANS-DABCYL Endopeptidase Substrates.

Selective expression of proteases and controlled proteolytic activity is a hallmark of developmental processes in biology.  While many proteases act on terminal amino acid sites (exopeptidases), most proteases cleave their substrates at internal positions of proteins (endopeptidases) that are sequence dependent.  Measurement of endopeptidase/protease activity is often accomplished by constructing Fluorescence Resonance Energy Transfer (FRET) peptides that contain an N-terminal fluorescence acceptor group, like 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a matched C-terminal fluorescence donor group, such as 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (M0273: EDANS).  The DABCYL group acts to quench the fluorescence of the EDANS fluorophore since the emission band of EDANS (490 nm) displays excellent overlap with the broad visible absorption band of DABCYL.  Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence can be further amplified by incorporation of a central Proline or Pro-Gly segment, which serves as a conformation hairpin bringing the two groups in close physical proximity.  Marker Gene provides the DABCYL quencher in a convenient reactive form (M1051: DABCYL-SE) that can be used to directly link to the amino terminus of most peptides and proteins.  A g-aminobutyric acid (GABA) spacer is also sometimes included between the amino terminus and the DABCYL group to prevent steric hindrance of the substrate binding from the bulky acceptor group.  Many FRET Protease Substrates have been constructed for a variety of important assays including HIV proteases (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg), (DABCYL-GABA-Ser-Val-Val-Tyr-Pro-Val-Val-Gln-EDANS),  Renin (Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg), Cathepsin B (DABCYL-Arg-Leu-Arg-Gly-Phe-Glu X ica Prom the bulky acceptor group.  inus and the DABCYL group to prevent steric hinderance  their substrate(EDANS), matrix metalloproteinase (DABCYL-Gaba-Pro-Gln-Gly-Leu-GIu(EDANS)-AIa-Lys-NH2), and papain  DABCYL-Leu-Arg-Gly-Phe-Glu(EDANS).  For more information about these assays, please visit our website or see the references below.  

• Wang G.T., Matayoshi, E., Huffaker, H.J., Krafft, G.A., (1990)."Design and Synthesis of New Fluorogenic HIV Protease Substrates Based on Resonance Energy Transfer." Tet. Lett 31: 6493
• Matayoshi E.D., Wang G.T., Krafft G.A., Erickson J. (1990) "Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer." Science 247: 954-958.
• Szollosi J, Damjanovich S, Matyus L. (1998) "Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research." Cytometry 34: 159-179.
• Pattanaik, P., Jain, B., Ravindra, G., N.Gopi, H.N., Pal, P.P., Balaram,H.,  Balaram P.,  (2003) “Stage-specific profilling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate.”  Biochemical and Biophysical Research Communications 309: 974-979. 
Capobianco J.O., Lerner C.G., Goldman R.C. (1992) "Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase." Anal. Biochem. 204: 96-102.
Holzman T.F., Chung C.C., Edalji R., Egan D.A., Martin M., Gubbins E.J., Krafft G.A., Wang G.T., Thomas A.M., Rosenberg S.H. (1991) "Characterization of recombinant human renin: kinetics, pH-stability, and peptidomimetic inhibitor binding." J. Protein Chem. 10: 553-563.
• Stachowiak, K., Tokmina, M., Karpiñska, A., Sosnowska, R., Wiczk W., (2004) “Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B” Acta Biochimica Polonica 51(1): 81-92.

Affordable Value at Marker Gene. 

Marker Gene has announced a price increase of 5% on most of our standard stock items, effective January 1, 2007.  We will still honor quotes and purchase orders for items placed before the end of the year, and also special pricing for customers who have standing orders with us.  There is still a 20% discount off the list price for orders of 5 or more units of any individual catalog item, as before.   Please contact our customer service department at 1-888-218-4062 for more information.  If you would like to place an order, please use our direct FAX lines: 1-541-342-1960 and 1-541-687-7963. 

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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