Marker Gene Monthly Newsletter   

April, 2004

Volume 4, Number 4

© 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.

TAMRA Protein/Antibody Labeling.

TAMRA is a bright red fluorescent labeling dye that has achieved prominence because of its high extinction coefficient, stability and pH insensitivity.  It is routinely used as a dye for oligonucleotide labeling, automated DNA sequencing, carbohydrate and protein labeling and has even been used for direct labeling of live cells.  Marker Gene has now developed a new TAMRA protein and antibody labeling kit that contains the highly reactive N-hydroxysuccinimidyl ester (SE) derivative of (5(6)-Carboxytetramethylrhodamine, M0962).  Detection of TAMRA can be easily observed via direct fluorescence measurement (EX 540 nm; EM: 565 nm) of labeled proteins or antibodies using classical methods or can be used with two fluorescent dyes in combination (i.e. fluorescein labeling: EX: 488nm; EM520 nm), via fluorescence resonance energy transfer (FRET).  The fluorescence of TAMRA conjugates can also be efficiently quenched using the new QSY 7® quencher.  Marker Gene now sells this popular fluorescent labeling kit (Product Number M0972).  For more information about the new TAMRA Protein/Antibody Labeling Kit and its use for conjugation of antibodies, proteins, carbohydrates and oligonucleotides, see our web site or the references below.

• Carol A Casey, Cheryl R Baldwin, Jacy L Kubik, Agnes M Hindemith and Benita L McVicker (2004)  “Use of Flow Cytometric Analysis to Examine the Uptake of Apoptotic Bodies by Healthy Hepatocytes” Comparative Hepatology 3 (Suppl 1): S40. 
•   Farinas J, Verkman AS. (1999) “Receptor-mediated targeting of fluorescent probes in living cells.” J. Biol Chem. 274(12): 7603-6.
• Oefner P.J., Huber C.G., Umlauft F., Berti G.N., Stimpfl E., Bonn G.K., (1994) "High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids." Anal Biochem 223, 39-46
• Zhao J.Y., Diedrich P., Zhang Y., Hindsgaul O., Dovichi N.J., (1994) "Separation of aminated monosaccharides by capillary zone electrophoresis with laser-induced fluorescence detection." J. Chromatogr. B Biomed. Appl. 657: 307-313
• Ravdin P, Axelrod D "Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization.". Anal. Biochem. 80: 585-592 (1977).
  

Retroviral Titer Estimated Using Marker Genes.

The measurement of retroviral titers can be a labor-intensive process, involving serial dilution of virus suspensions, and many days of cell culture. Alternately, if a RV vector containing a marker gene is used, such as the lacZ gene or the green fluorescent protein gene (gfp), viral titers can be estimated with significant accuracy by simple staining or fluorescence spectroscopy.  The viral titer is often referred to as colony-forming units (CFUs). When using a marker gene and a fluorogenic substrate like fluorescein di-b-D-galactopyranoside (FDG, M0250), the number of viral particles capable of forming individual green cells per unit volume is defined as marker-forming units (MFU). After adjusting for time factors, MFU units have been found to be comparable to CFU in most cases.  The use of b-galactosidase as a reporter in retroviral vectors has a number of advantages over other reporters. The enzymatic activity of b-galactosidase can also be used as a quantitative measure of gene expression from the viral promoter in a cell line of choice, the signals are higher than with GFP, and FACS analysis can be performed on infected cells following incubation with a substrate like fluorescein di-b-D-galactopyranoside, resorufin-galactoside (M0203), or 3-Carboxyumbelliferyl b-D-Galactopyranoside (CUG, M0257).  For more information about using marker genes for measuring retroviral titers, see our web site or the references below.

• “Systematic comparison of a color reporter gene and drug resistance genes for the determination of retroviral titers.” (1999) Sapp CM; Li T; Zhang J., J. Biomed. Sci . 6(5): 342-8.
• “Oncolytic viral therapy for human ovarian cancer using a novel replication-competent herpes simplex virus type I mutant in a mouse model.” (2003) Nawa A ; Nozawa N ; Goshima F.,  Nagasaka T.,  Kikkawa F.,  Niwa Y.,  Nakanishi T.,  Kuzuya K.,  Nishiyama Y.,  Gynecol. Oncol . 91(1): 81-8.
• “The use of recombinant adeno-associated viral vectors for the transduction of epithelial tumor cells.” (1997) Hoerer M., Bogedain C.,  Scheer U., Heberger C.,  Steyrer S.,  Burger A.,  Maass G.,  Int. J. Immunopharmacol. (1997) 19(9-10): 473-9.
• “A high-throughput hybridization method for titer determination of viruses and gene therapy vectors” E. Morrey Atkinson, Daryn J. Debelak , Lori A. Hart , Thomas C. Reynolds Nucl. Acids. Res. 26 (11): 2821.

Toward MRI Imaging of lacZ in vivo.

Gene therapy continues to hold great promise for disease treatment.  However, it is often difficult to assess transfection efficiency spatially and temporally inside living tissues.  Many fluorescent and chemiluminescent gene expression systems have been developed by Marker Gene and others, but these have difficulty when used in vivo, due to the opaqueness of living tissues.  Two recent MRI imaging techniques have been developed to help with tissue imaging using MRI (magnetic resonance imaging) detection.  A fluorinated derivative of ONPG, 4-Fluoro-2-nitrophenyl-beta-D-galactopyranoside (PFONPG) has shown promise as a novel prototype NMR-sensitive molecule for transfected cells in culture.  The molecule is stable in solution and with respect to wild-type cells, but the enzyme causes liberation of the fluorophenol, accompanied by a ¹⁹F-NMR chemical shift of 5-10 ppm, dependant on pH.   The released phenol, however, is quite toxic and also quickly leaks from cells with lacZ activity.  The second involves the use of an MRI contrast agent containing a gadolinium caged structure in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a galactoside substrate that can be removed by galactosidase enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal.  Although gadolinium is a highly toxic heavy metal ion, and the caged compound has poor cell permeability, it has found use in live animal studies.  Despite the shortcomings of these prototype systems, new reagents for use detecting marker gene activity inside living tissues are on the horizon.  For more information about these approaches and others, see our website or the references below:

• “Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase. “ (2004) W. Cui, P. Otten Li Y, Y. Li, K. S.Koeneman, J. Yu, and R. P.Mason, Magn. Reson. Med .  51(3): 616-20.
• “In vivo visualization of gene expression using magnetic resonance imaging. (2000) Louie AY ; Hüber MM ; Ahrens ET ; Rothbächer U ; Moats R ; Jacobs RE ; Fraser SE ; Meade TJ., Nat. Biotechnol. 18(3): 321-5.

Transgenic versus Endogenous Enzyme Analysis.

Gene therapy continues to hold great promise for disease treatment.  However, it is often difficult to assess transfection efficiency spatially and temporally inside living tissues.  Many fluorescent and chemiluminescent gene expression systems have been developed by Marker Gene and others, but these have difficulty when used in vivo, due to the opaqueness of living tissues.  Two recent MRI imaging techniques have been developed to help with tissue imaging using MRI (magnetic resonance imaging) detection.  A fluorinated derivative of ONPG, 4-Fluoro-2-nitrophenyl-beta-D-galactopyranoside (PFONPG) has shown promise as a novel prototype NMR-sensitive molecule for transfected cells in culture.  The molecule is stable in solution and with respect to wild-type cells, but the enzyme causes liberation of the fluorophenol, accompanied by a ¹⁹F-NMR chemical shift of 5-10 ppm, dependant on pH.   The released phenol, however, is quite toxic and also quickly leaks from cells with lacZ activity.  The second involves the use of an MRI contrast agent containing a gadolinium caged structure in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a galactoside substrate that can be removed by galactosidase enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal.  Although gadolinium is a highly toxic heavy metal ion, and the caged compound has poor cell permeability, it has found use in live animal studies.  Despite the shortcomings of these prototype systems, new reagents for use detecting marker gene activity inside living tissues are on the horizon.  For more information about these approaches and others, see our website or the references below:

• “Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase. “ (2004) W. Cui, P. Otten Li Y, Y. Li, K. S.Koeneman, J. Yu, and R. P.Mason, Magn. Reson. Med .  51(3): 616-20.
•  “In vivo visualization of gene expression using magnetic resonance imaging. (2000) Louie AY ; Hüber MM ; Ahrens ET ; Rothbächer U ; Moats R ; Jacobs RE ; Fraser SE ; Meade TJ., Nat. Biotechnol. 18(3): 321-5.
  
  

Depurination and DNA Strand Breakage.

Depurination of DNA results in the loss of a purine from the DNA backbone and DNA strand breakage.  Such DNA lesions are often the result of ionizing radiation, free radicals, or alkylating reagents that destabilize the N-glycosidic bond.  Left unchecked, they have the potential to cause mutagenesis and carcinogenesis.  Depurination is also a well-known side reaction in DNA synthesis and in isolation of DNA or RNA samples from gels, when the oligonucleotide is subjected to weak acid conditions.  During synthetic  deprotection of trityl protecting groups, depurination can cause chain cleavage producing fragmentation.  Depurination is especially troublesome in oligonucleotides that are either long or A-rich.  Upon release of the base (purine), a reducing sugar (ribose) is left on the backbone.  Analysis of these abasic sites can take advantage of the aldehyde thus produced to quantitate depurination.  These include reactive biotin (ARP) or fluorescein (FARP) labeling compounds which incorporate a carboxymethylhydroxylamine function.  Marker Gene is developing new labeling reagents for depurination detection, with improved sensitivity and stablity.  For more information about these techniques, see the references below. 

• T. Suzuki, S. Ohsumi, K. Makino “Mechanistic studies on depurination and apurinic site chain breakage in oligodeoxyribonucleotides.” (1994) Nucleic Acids Res.  22 (23): 4997–5003.
• Ide H., Akamatsu K., Kimura Y., Michiue K., Makino K.,  Asaeda A.,  Takamori Y., Kubo K., (1993) “Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.” Biochemistry 32(32): 8276-83.
• Makrigiorgos G.M., Chakrabarti S., Mahmood A. (1998) “Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.” Int J Radiat Biol 74(1): 99-109.

2004-2005 Catalog Will Be Available Soon.

The 2004-2005 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.
Sign up now!

 

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. 

To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com.

To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.