Marker Gene Monthly Newsletter   

August, 2004

Volume 4, Number 8

© 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.

Intracellular GFP Localization using Fusion Proteins.

A variety of new methods have been developed for intracellular localization of co-expressed marker genes including GFP (green fluorescent protein), CAT, amp, or lacZ to allow  accumulation inside subcellular organelles within living cells.  For example, although GFP is usually localized in the cytoplasm, new amino- and carboxy- terminal GFP (green fluorescence protein) fusions have been developed that can provide localization to a variety of intracellular structures including to the nucleus (using an SV40 T-antigen nuclear localization signal), to the mitochondria (using a targeting sequence from subunit VIII of cytochrome c oxidase), to microtubules (human a-tubulin fusion) or to actin filaments (human b-actin fusion).  Similar methods have been used in plants for subcellular localization of reporter gene expression.  In addition, cDNA libraries are being used to map potential intracellular localization motifs by using similar marker gene - fusion protein methods.  For more information about these exciting new techniques, please see our web site or the references below.

• Simpson J.C., Wellenreuther R., Poustka A., Pepperkok R., Wiemann S., EMBO Reports (2000) 1:287-292
• Khodjakov A., Cole R.W., Rieder C.L.,  (1997) Cell Motil. Cytoskeleton 38:311–317.
• Roberts, P. M. & Goldfarb, D. S. (1998) Methods Cell Biol. 53:545–547.
• Living Colors Subcellular Localization Vectors (1998) CLONTECHniques XIII(4):8–9.
• Spergel D.J., Krüth U., Shimshek D.R., Sprengel R., Seeburg P.H., Prog Neurobiol (2001) 63(6): 673-86
• Kanazawa M., Yano M., Namchai C., Yamamoto S., Ohtake A., Takayanagi M., Mori M., Niimi H.,  (1997) Biochem. Biophys. Res. Commun. 239:580–584.
  

Nanoparticles for in vivo Marker Gene Delivery.

The potential of using nanoparticles to deliver gene expression vectors in vivo, is being developed by a number of laboratories world-wide.  For example, poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, has been used to prepare recombinant TK (thymidine kinase) plasmid P(EGFP-AFP) nanoparticles that also contain the GFP protein to hepatocarcinoma cells. Similarly, a Hepatitis B virus envelope L particle, which contains a hollow nanoparticle core, was recently used for efficient and specific transfer of the gene for green fluorescent protein (GFP) into human hepatocytes both in culture and in a mouse xenograft model.  A number of other nanoparticles have also been developed for gene transfer including silica based particles, magnetic particles, gelatin/albumin/polyalkylcyanoacrylate and chitosan based materials.  The potential of these techniques for use in gene therapy and drug delivery applications is very promising.  For more information on these new methods, please see the references below:

• Yamada T., Iwasaki Y., Tada H., Iwabuki H., Chuah M.K., VandenDriessche T., Fukuda H., Kondo A., Ueda M., Seno M., Tanizawa K., Kuroda S Nat Biotechnol  (2003) 21(8): 885-90.
• He Q., Liu J., Sun X., Zhang Z.R., World J Gastroenterol (2004) 10(5): 660-3.
• Chen Y., Xue Z., Zheng D., Xia K., Zhao Y., Liu T., Long Z., Xia J Curr Gene Ther (2003) 3(3): 273-9.
• Ravi Kumar M., Hellermann G., Lockey R.F., Mohapatra S.S., Expert Opin Biol Ther (2004) 4(8): 1213-24.

New lacZco Vectors Now Available.

Marker Gene has collaborated with Dr. Donald Anson and coworkers at the Department of Chemical Pathology, Women's and Children's Hospital in North Adelaide, Australia to develop several new lacZ expression vectors with high expression efficiency in mammalian cells.  The coding sequence for the E. coli beta-galactosidase gene was codon-optimized and when expressed in mammalian cells this results in the expression of beta-galactosidase at levels 15-fold higher than those from an analogous construct containing the native E. coli gene sequence.  RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct, the codon-optimized gene gave an approximately five-fold increase in apparent titre, as determined by X-Gal staining, in comparison to an analogous construct containing the native E. coli gene.  In addition, codon-optimization resulted in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimized gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells, the codon-optimized beta-galactosidase gene has a number of advantages over the native E. coli gene sequence.  For more information on this new vector system, see the references below.  New vectors soon to be available are 1) pCMVb lacZco (Product M1015); 2) pSV40b lacZco (Product M1016); 3) pCMVb lacZ NLS12co (Product M1017); and 4) pSV40b lacZ NLS12co (Product M1018).  See our website or contact us for more information on ordering these new vectors from Marker Gene soon!

• Anson D.S., Limberis M., J Biotechnol (2004) 108(1): 17-30.
• http://biology.kenyon.edu/courses/biol09/betagal/FRAMES/
• MacGregor G. R., Caskey C. T., Nucleic Acids Res. (1989) 17:2365.
• Okayama H., Berg P., Mol. Cell Biol. (1983) 3:280-289.
• MacGregor G. R., et al., Somat. Cell Mol. Genet. (1987) 13:253-265.

New Apoptosis Pathways in Plants.

Programmed cell death (PCD, apoptosis) is a well-known process in mammalian cells, and exhibits both beneficial as well as detrimental activities.  It can serve to remodel tissues during development and when organisms respond to environmental challenges, but can also produce degeneration and other unwanted effects. In virally infected plants, infected cells are killed by programmed cell death during the hypersensitive response. However, the signature caspases that promote programmed cell death found in animals have not been found in plants. Dr. Horiyki Hatsugai and co-workers at Kyoto University in Japan havenow identified VPE, a vacuolar processing enzyme that may be a component of programmed cell death in tobacco plants. This enzyme is confined within a vacuole inside the plant cell wall.  VPE is a cysteine protease that was originally found to be involved in maturation of seed storage proteins, and also has found to have activity in leaf senescence, lateral root formation, and cell death.  It appears to have a caspase-1 like activity, and is inhibited by the caspase-1 inhibitor Ac-YVAD-CHO as well as the VPE inhibitor Ac-ESEN-CHO.   The ability of these inhibitors of the PCD pathway in tobacco plants challenged with the hypersensitive response caused by pathogens (i.e. tobacco mosaic virus) was demonstrated.  A similar VEIDase activity was also recently identified in plants by Dr. Peter.Bozhkov and co-workers at the Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences. For more information about these new pathways in plants, see the references below:

• Hatsugai N.,  Kuroyanagi M., Yamada K., Meshi T., Tsuda S., Kondo M., Nishimura M., Hara-Nishimura I., Science (2004) 305(5683): 855-858.
• Hoeberichts F. A., ten Have A., Woltering E.J., Planta (2003) 217: 517.
• Bozhkov P.V., Filonova L.H., Suarez M.F., Helmersson A., Smertenko A.P., Zhivotovsky B., von Arnold S., Cell Death Differ (2004) 11: 175.
  
  

Serum Lysophosphatidic Acid (LPA) Assay.

Ovarian cancer is usually diagnosed when the cancer has already progressed to Stage 3 or 4.  Recently, evaluated levels of Lysophosphatidic Acid (LPA) have been found to be a more reliable measure of the disease in ovarian cancer patients versus the level of CA-125, a common biomarker for ovarian cancer.  Almost all healthy women were found to have low levels of lysophospholipids, but most ovarian cancer patients were found to have high levels of these substances making them an accurate predictor of ovarian cancer with a low "false positive" (lower than 4%).  In most of these studies, LPA levels were determined using chromatography/mass spectroscopy.  Lysophosphatidic acid can also be determined by measuring serum LPA concentration using a dot immunogold filtration assay (DIFA) based upon a polyclonal antibody binding to LPA, or by using a combined enzymatic, colorimetric assay method, and measuring H₂O₂ release.  LPA is produced from phosphatidyl choline by the action of two phospholipase enzymes.  For more information about these new early detection markers, see our website or the references below:

• Sutphen R., Xu Y., Wilbanks G.D., Fiorica J., Grendys E.C. Jr, LaPolla J.P., Arango H., Hoffman M.S., Martino M., Wakeley K., Griffin D., Blanco R.W., Cantor A.B., Xiao Y.J., Krischer J.P., Cancer Epidemiol Biomarkers Prev. (2004) 13(7): 1185-91.
• Kishimoto T., Matsuoka T., Imamura S., Mizuno K., Clin. Chim. Acta (2003) 333(1): 59-67.
• Kishimoto T., Soda Y., Matsuyama Y., Mizuno K., Clin. Biochem. (2002) 35(5): 411-6.
• Chen X., Yang X.Y., Wang N.D., Ding C., Yang Y.J., You Z.J., Su Q., Chen J.H., Scand. J. Clin. Lab. Invest. (2003) 63(7-8): 497-503
•   Xu Y., Shen Z., Wiper D.W., Wu M., Morton R.E., Elson P., Kennedy A.W., Belinson J., Markman M., Casey G., JAMA (1998) 280(8): 719-23.
• Shen Z., Wu M., Elson P., Kennedy A.W., Belinson J., Casey G., Xu Y., Gynecol Oncol. (2001) 83(1): 25-30.
  
  

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.