Marker Gene Monthly Newsletter   

September, 2004

Volume 4, Number 9

© 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.

Improved b-galactosidase detection in lacZ transfected cells.

The b-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of co-transfected genes. Fluorogenic substrates allow sensitive detection of enzyme activity in viable cells, which, subsequently, can be isolated for further study on the basis of their fluorescence emission using FACS.  Several common fluorogenic substrates have been used for their intracellular staining ability (FDG (M0250), Resorufin Galactoside (M0203) or CUG (M0255)), all of which produce the fluorophores upon enzyme activity.  The intensity of their fluorescent signal and selectivity toward transfected b-galactosidase activity vs. endogenous lysosomal enzyme activity varies.  Attempts to improve selectivity by raising intracellular lysosomal pH with compounds such as chloroquine, which provide a suboptimal pH for endogenous enzyme activity, often leads to significant cell death. Recently, inhibition of dye efflux using the drug  verapamil has been found to enhance the selectivity of these substrates toward the lacZ-encoded enzyme activity by approximately two- to threefold.  Verapamil is a calcium ion influx inhibitor which tends to block membrane transport by efflux proteins.  Incubation with another efflux protein inhibitor, probenecid, on the other hand, has thus far shown only a slight improvement of fluorescence retention at 37^oC.  Typical working concentrations for these new inhibitors are either 1 mM probenicid or 100 µM verapamil when using 50 mM FDG.  The potential use of these compounds for improved detection of lacZ transfected cells or for use in other studies (i.e. multidrug resistance assays) is ongoing.  Please see our website or the references below for more information about these new techniques.

• Poot M., Arttamangkul S., (1997) "Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells." Cytometry 28(1): 36-41.
• Rosati A., Candussio L., Crivellato E., Klugmann FB., Giraldi T., Damiani D., Michelutti A., Decorti G., Bodipy-FL-verapamil: a fluorescent probe for the study of multidrug resistance proteins. Cell Oncol (2004) 26(1-2): 3-11.
• Raaijmakers H.G., Van Den Bosch G., Boezeman J., De Witte T., Raymakers R.A. (2002) “Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors.”  Cytometry.  49(4):135-42.
  

Nonviral Gene Transfer and Targeting in vivo.

A new approach to non-viral gene transfer encapsulates plasmid DNA inside polyethyleneglycol immunoliposomes (PILs).  The PIL gene transfer technology has been used in adult mice, rats, and rhesus monkeys. Non-viral plasmid DNA is encapsulated in the interior of an 85-nm liposome, and the surface of the liposome is conjugated with several thousand strands of 2000 Da poly(ethylene glycol) (PEG). This pegylation of the liposome stabilizes the nanocontainer in vivo, and allows for prolonged blood residence time. In addition, the PEG liposome can be further modified for targeting to individual tissues or  across biological barriers (i.e the blood-brain barrier) using targeting ligands that are conjugated to the tips of  the PEG strands. The targeting ligand may be an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) that targets a cell surface receptor, and enables transport of the PIL across the membrane barrier.  Some of the targeting systems used include the transferrin receptor (TfR), expressed at both the BBB and the brain cell membrane and the human insulin receptor (HIR), which enables global gene expression within the brain following an intravenous injection. The exogenous gene is expressed in brain 24–48 h after gene administration.  For more information about these new techniques please see our website or the references below.

• Shi N., ZhangY., Boado R.J.,,Zhu C., PardridgeWM.”Brain-specific expression of an exogenous gene after i.v.administration.Proc Natl Acad Sci U S A 2001; 98: 12 754–12 759.
• Shi N, Boado RJ, Pardridge WM. (2001) “Receptor-mediated gene targeting to the tissues in the rat in vivo.” Pharm Res 18: 1091–1095.
• Zhang Y., Schlachetzki F., Pardridge W.M., (2003) “Global non-viral gene transfer to the primate brain following intravenous administration.” Mol. Ther.  7: 11–18.
• Pardridge W.M., (2003) “Gene targeting in vivo with pegylated immunoliposomes.” Methods Enzymol. 373: 507–528.
• Maruyama K, Takahashi N, Tagawa T, Nagaike K, Iwatsuru M. ”Immunoliposomes bearing polyethyleneglycol-coupled Fab' fragment show prolonged circulation time and high extravasation into targeted solid tumors in vivo.” FEBS Lett. (1997) 413(1):177-80.

Mitochondrial Functional Analysis.

Screening methods for identifying cultured cells with deficient mitochondrial function often use the ultrasensitive substrate (dihydrorhodamine 123, M0545) (DHR123), an uncharged, nonfluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining is observed in cells that respire normally.  Fluorescence however, is significantly reduced in cells with mitochondrial respiratory chain dysfunction.  Numerous studies show that DHR123 is useful for assessing mitochondrial function in single cells, and can also be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures. Artificial induction of an oxidative burst in cells can be accomplished using compounds like the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP), the protein kinase C ligand phorbol 12-myristate-13-acetate (PMA) or bacterial challenge.  For more information about these techniques, see our website or the references below.

• SobreiraC ; Davidson M ; King MP ; Miranda AF (1996) “Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured cells harboring mitochondrial DNA mutations.”J Histochem Cytochem  44(6): 571-9.
• Rothe G, Oser A & G. Valet. (1988) Dihydrorhodamin 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 75: 354 - 355.
• Dobmeyer, T.S., Raffel, B. Dobmeyer, J.M., Findhammer, S., Klein, S.A., Kabelitz, D. Hoelzer, D., Helm, E.B. & Rossol.(1995) Decreased function of monocytes and granulocytes during HIV-1 infection correlates with CD4 cell counts. Eur. J. Med. Res. 1: 9-15.
• Gessler, P., Nebe, T. Birle, A., Haas, N. & W. Kachel. (1996) Neutrophil respiratory burst in term and preterm neonates without signs of infection and in those with increased levels of C-Reactive Protein. Pediatr. Res. 39: 843-848.
• Elbim, C., Chollet-Martin, S., Bailly, S., Hakim, J. & M.A. Gougerot-Pocidalo. (1993) Priming of polymorphonuclear neutrophils by tumor necrosis factor in whole blood: Identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides. Blood 82: 663-640.

New GUS Systems 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. Marker Gene now provides several new products for monitoring cloned GUS activity in live plant cells and in plant extracts, including the ß-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877) and the new cell permeant substrate fluorescein di-glucuronide, di-methyl ester (M0969).The M0877 kit provides all the reagents, buffers, and a detailed protocol for easy quantitative measurement 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. Please see our Web site or the references below for more information:

• Kain, S.R., Ganguly, S., Current Protocols in Molecular Biology. (1996) p. 9.6.1.
• Jefferson R.A., Burgess S.M., Hirsh D.,  (1986) “beta-Glucuronidase from Escherichia coli as a gene-fusion marker.” Proc. Natl. Acad. Sci. USA. 83(22): 8447-51.
• Jefferson R.A., Kavanagh T.A., Bevan M.W., (1987)  “GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.” EMBO J. 6: 3901-3907.
• Kosugi S., Suzuka I., Ohashi Y., Murakami T., Arai Y.,  (1991) “Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants.” Nucleic Acids Res 19(7): 1571-6.
  
  

2005 Catalog Will Be Available Soon.

The 2005 edition of the Marker Gene catalog is in production.  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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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

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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