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Marker Gene Monthly Newsletter   

December, 2004

Volume 4, Number 12

 

© Copyright MGT, Inc., 2004.  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. 



Somatic Hypermutation to Produce New Fluorescent Proteins.


Because cells and tissues absorb light below about 600 nm, long-wavelength fluorescence excitation and emission are important for tissue or whole animal analysis of gene expression.  For this reason, Dr. Roger Tsien and colleagues at the Howard Hughes Medical Institute and the University of San Diego are attempting to harness B-cells and use them to make mutant fluorescent proteins with new longer-wavelength emission colors.  The immune system has designed B-cells with the ability to hypermutate in order to produce many clones of different antibodies to ward off infection.  This process, called somatic hypermutation takes place in germinal centers formed in the secondary lymphoid tissues.  B-cells proliferating in these germinal centers experience random mutations in the genes encoding the variable region of their immunoglobulin molecules and are subsequently selected for high-affinity binding to antigen.  Tsien’s group has cloned a red fluorescent protein (RFP) into human B-cells and allowed them to make variants that could then be isolated based on their colors by flow-cytometry.  They found several new long-wavelength RFP’s (EM ~650 nm) and isolated the resulting proteins.  For more information about these new techniques for isolation of altered wavelength fluorescent proteins or for production of other mutant proteins, see the references below or visit our website.




·         L. Wang , W. C. Jackson , P. A. Steinbach , R. Y. Tsien “Evolution of new nonantibody proteins via iterative somatic hypermutation” (2004) PNAS 101(48): 16745-16749.

·          C. L. Wang , R. A. Harper, M. Wabl (2004) “Genome-wide somatic hypermutation” PNAS 101(19): 7352-7356.

·          Shen, H. M., Peters, A., Baron, B., Zhu, X. & Storb, U. (1998) “Mutation of BCL-6 Gene in Normal B Cells by the Process of Somatic Hypermutation of Ig Genes” Science 280: 1750–1752.

·         Gordon, M. S., Kanegai, C. M., Doerr, J. R. & Wall, R. (2003) “Somatic hypermutation of the B cell receptor genes B29 (Igb, CD79b) and mb1 (Iga, CD79a)”Proc. Natl. Acad. Sci. USA 100: 4126–4131.

·         R. Richards-Kortum, E. Sevick-Muraca (1996) “Quantitative Optical Spectroscopy for Tissue Diagnosis“ Annu. Rev. Phys. Chem. 47: 555–606.

aGalA Gene Therapy Localization in Fabry disease

Researchers at the University of Toronto and the Ontario Cancer Institute at the Princess Margaret Hospital have developed a new early stage gene therapy protocol that shows promise for sustained correction of Fabry's disease, a rare, inherited metabolic disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (aGalA).   Without the enzyme, the lipid globotriaosylceramide (Gb3) accumulates in the vascular epithelium, heart, kidneys, cornea, and other tissues, causing cardiovascular disease, renal failure, corneal opacities, angiokeratoma, paresthesias, and hypohidrosis.  An engineered lentiviral vector administered a day or two after birth was used to correct the defect in mice, ensuring they produce the appropriate enzyme at relevant levels (greater than 5% normal expression levels).  The early gene transfer was used to alleviate immune recognition of the foreign gene.  In a parallel study, Medin and his colleagues were able to monitor gene therapy vector activity in treated live mice at various points in their lives over a significant time-span using a luciferase expression and CCD camera imaging.  They tracked whole body luciferase activity in various individual organs, including the brain.  These findings have special relevance in Canada. There is a significant concentration of Fabry patients in Nova Scotia, all descended from an immigrant who arrived there in the late 1600s.  Presently, the only corrective treatment option is repeated recombinant enzyme infusion.  For more information about these new techniques, please see the following references, or visit our website.

  • Makoto Yoshimitsu, Takeya Sato, Kesheng Tao, Jagdeep S. Walia, Vanessa I. Rasaiah, Gillian T. Sleep, Gary J. Murray, Armando G. Poeppl, John Underwood, Lori West, Roscoe O. Brady, and Jeffrey A. Medin (2004) “Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors” PNAS  101: 16909-16914.
  • Medin, J.A., Tudor, M., Simovitch, R., Quirk, J.M., Jacobson, S., Murray, G.J., Brady, R.O. (1996) “Correction in trans for Fabry disease: Expression, secretion, and uptake of a-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.” Proc Natl Acad Sci USA 93: 7917-7922.
  • Mignani R., Cagnoli L., (2004) “Enzyme replacement therapy in Fabry's disease: recent advances and clinical applications.” J. Nephrol. 17(3): 354-363.

Choosing Optical Filter Sets for Fluorescence Applications.

Choosing the right filters for your fluorescence application is sometimes a complicated endeavor, mostly because there are many optical filter companies and various wavelength filters available.  The primary filtering elements in a reflected light fluorescence microscope are typically a set of three filters housed in an optical block, commonly referred to as a fluorescence filter cube or filter block. The cube will contain an excitation and emission filter in combination with a dichromatic beamsplitter (often termed a dichroic mirror) to separate excitation illumination from the weak secondary fluorescence emitted by the specimen.  Most flow cytometers or microtiterplate readers have special filters available from the manufacturers.  But these filters use the same mechanism; an excitation filter which allows light into the sample at wavelengths up to the excitation maximum and an emission filter, typically a bandpass filter, which allows all light above a certain wavelength (typically the emission maximum wavelength).  The dichroic mirror is typically not necessary for non-microscopic analysis.  Marker Gene has a list of common filter sets and their corresponding fluorescence applications on our website at http://www.markergene.com/Filter Sets.htm .  More specific information or technical assistance can be obtained by calling or contacting our technical assistance staff at techservice@markergene.com 

·         Lowy, R.J., (1995) “Evaluation of triple-band filters for quantitative epifluorescence microscopy.” J. Microscopy 178 (3): 240-250.

·         Gundlach H., (2001) “Digital microscopy for multiparameter FISH imaging.” Anal. Quant. Cytol. Histol.  23(4): 268-272.

·         Tsurui H., Nishimura H., Hattori S., Hirose S., Okumura K., Shirai T., (2000) “Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.” J. Histochem. Cytochem. 48(5): 653-662.

A "Read-Write-Erase" Fluorescent Protein - Dronpa.

 

FRAP (fluorescence recovery after photobleaching) has long been used to track protein movements inside living cells and tissues.  But such systems are slow, and their ability to be reused repeatedly is limited. Recently Dr. Atsushi Miyawaki and colleagues at the Laboratory for Cell Function and Dynamics, RIKEN Brain Science Institute in Japan have engineered a new fluorescent protein from the coral Pectiniidae to be monomeric and to be repeatedly highlighted and erased by activation at 405 nm and 488 nm, respectively, without appreciable photobleaching. These properties allow such fluorescent proteins to be used for intracellular protein tracing repeatedly within individual cells. The cDNA encoding the protein was cloned into E. coli and also mammalian cells (HeLa cells) by cloning into a pcDNA3 vector. The system has been used to monitor both nuclear import and export of a protein kinase (ERK) using a fusion protein approach. The protein also has potential as an information storage medium with the ability to record, erase, or reread information nondestructively.  For more information about these exciting new techniques, please see the references below or visit our website.

  • R. Ando, H. Mizuno, A. Miyawaki (2004) "Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting" Science, 306: 1370-1373.
  • Irie M., Fukaminato T., Sasaki T., Tamai N., Kawai T., (2002) "Organic chemistry: A digital fluorescent molecular photoswitch" Nature 420(6917): 759-760.
  • Patterson, G.H. and Lippincott-Schwartz, J. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 297: 1873-1877 (2002).
  • Ando, R. et al. An Optical Marker Based on the UV-Induced Green-to-Red Photoconversion of a Fluorescent Protein. Proc. Natl. Acad. Sci. USA 99: 12651-12656 (2002).

Live Cell Viral Entry Assay Using Luciferase.

Several new assays have been developed to monitor viral entry into cells.  Although PCR or GFP fusion protein systems have been useful, they cannot identify active (replicating) viral particles from non-active virus.  Recent work from the laboratory of Dr. Robert Davey at the Department of Microbiology & Immunology, University of Texas Medical Branch has employed a novel luciferase C-terminal fusion to a viral envelope protein that targets the marker gene to the viral lumen.  Only when the incorporated luciferase is released from the viral lumen can it gain access to the substrate D-luciferin and light is emitted and readily detected.  When cells are perfused with D-luciferin, quantitative measurements of entry can be made in real time on live cells.  In addition, the total amount of viral infection can be determined by treating the same cells with detergent (1% NP-40) that exposes the luciferase encapsulated in the viral capsids.  Detailed kinetic examination of the entry mechanism of viruses and the discovery of novel entry inhibitors will be important uses of these new assays.  For more information about these assays or conditions for Live Cell Luciferase Detection, please see our website or the references below.

·        Kolokoltsov, R. A. Davey, (2004) “Rapid and Sensitive Detection of Retrovirus Entry by Using a Novel Luciferase-Based Content-Mixing Assay” J. Virology 78(10): 5124-5132.

·          Arnone M.I., Dmochowski I.J., Gache C., (2004) “Using reporter genes to study cis-regulatory elements.” Methods Cell Biol. 74: 621-52.

·         Cavrois, M., C. De Noronha, and W. C. Greene, (2002) “A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes.” Nat. Biotechnol. 20: 1151–1154.

New 2005 Catalog Now Available On-Line.

 

The 2005 edition of the Marker Gene catalog is now available on our website or by using the link www.markergene.com/catalog2005-2006.pdf .  Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products.  Be sure to add your name to our mailing list.  Please visit our Web site and fill out our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you.

 

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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 UO 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.
  • Enzyme substrate synthesis / coupled assay design.
  • Effector, Agonist, Antagonist, Inhibitor design, synthesis, and in vitro assays.
  • Chromogenic, Chemiluminescent, and Fluorescence Based Technologies.
  • New selection, effector and reporter uses for marker genes in Research, Cell Culture, Diagnostics and Therapeutics.
  • 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; (541) 342-3760; (541) 912-5320 or FAX us at (541) 342-1960 or you can write to us at 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

 

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