Marker Gene Monthly Newsletter   

June, 2006

Volume 6, Number 6

© 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.

DNA Transfection Using Dendrimers.

Transfection is routinely used for the introduction of plasmid DNA, messenger RNA (mRNA), short interfering RNA (siRNA), or microRNA (miRNA) molecules into cultured or primary cells or tissues. Transfection has become a common method for modulating gene expression, studying gene function, mapping and mutational analysis, or protein production. Researchers have used a variety of carrier molecules to transfer non-viral genes into cells of interest.  But, there is no single transfection reagent that can be applied to all types of cells and transfection efficiencies can vary depending on which cell type or reagent is used. 

Probably the most common transfection method for mammalian cells involves the use of liposome mediated DNA-delivery.  These protocols make use of synthetic cationic lipids such as N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA). Small monolayer liposomes containing DOTMA lipids will interact spontaneously with DNA to form lipid-DNA complexes with nearly 100% entrapment of the DNA. These liposome-DNA complexes will facilitate fusion of the complex with the plasma membrane of cells, resulting in both uptake and expression of the DNA.

Recently, polyamine dendrimers have been developed for use in high efficiency transfection of cells and tissues.  Dendrimers are highly branched, three-dimensional globular (spherical) macromolecules that are capable of condensing DNA into small compact complexes that increase plasmid transfection efficiency by improving cell membrane permeability as well as DNA targeting and stability. DNA-dendrimer complexes typically maintain a net positive charge that allows them to bind to negatively charged receptors on the cell surface (e.g. phospholipids, sialylated glycoproteins or glycolipids on the cell surface of eukaryotic cells).  These then become internalized by endocytosis.  Once inside, the dendrimer-DNA complex is targeted to lysosomes after fusion with the endosome, and the dendrimers release the DNA.  The dendrimers also seem to help lower lysosomal pH and subsequently inhibit lysosomal nucleases leading to improved DNA stability.  The dendrimer–DNA complexes are even known to promote entry into the nucleus.  Dendrimers are typically quite stable in vivo and are not temperature, enzymatic or pH sensitive, providing high transfection efficiency in most tissue culture models.  However, as a result, dendrimers are also not biodegradable and can lead to significant cytotoxicity in some transfection host cell lines.  Marker Gene is currently working with our distributors to bring you several high-efficiency starburst-type polyethyleneimine (PEI) dendrimers for use in efficient transfection protocols.  For more information about these new reagents and applications, please visit our website or see the references below.

•  Felgner, P.L., Gadek,T.R.,  Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen M., (1987) “Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure” Proc. Natl. Acad. Sci.  84(21): 7413-7417.
• Tang, M.X., Redemann, C.T. and Szoka, Jr., F.C. (1996) ”In vitro gene delivery by degraded polyamidoamine dendrimers.”  Bioconjugate Chem. 7: 703.
• Luo, D., Saltzman W.D. (2000) “Synthetic DNA delivery systems” Nat. Biotechnol. 18: 33-37.
• Pollard, H. et al. (1998) “Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.” J. Biol. Chem. 273: 7507–7511.
  

Long-Wavelength Phosphatase Analysis.

There are a variety of phosphatase enzymes important for regulation of protein activity, cell metabolism, signal transduction or in regulation of various biochemical pathways in vivo.  Many of these phosphatases act by removing phosphate groups from DNA (5'phosphate) or by action on phosphotyrosine or phosphotheonine residues of proteins.  The best known of the non-metalloenzymes are the protein tyrosine phosphatases, which hydrolyse phosphotyrosine residues. However, the metalloenzymes by far comprise the greatest bulk of phosphatases in the cell, and contain such enzymes as alkaline phosphatase, the serine threonine phosphatases and inositol monophosphatase.  The change in alkaline phosphatase levels has been implicated in a variety of physiological and pathological events, such as bone development, bone-related diseases, gestation related diseases, inflammatory bowel disease, post-parathyroidectomy syndrome, as well as in drug metabolism and toxicity.  Alkaline phosphatase is also a popular enzyme used for conjugation with secondary antibodies for immunoassays and ELISA-based diagnostic methods as well as immunohistochemical staining techniques and Northern, Southern and Western blot analyses. In addition, a high level of expression of alkaline phosphatase has been found to characterize the undifferentiated state of the embryonic stem (ES) cell.  Endogenous alkaline phosphatase has therefore recently been established as a marker for embryonic stem (ES) cells, making it ideal for routine verification of pluripotency in ES cells and assessment of overall in vitro stem cell pluripotency.  In addition, alkaline phosphatase treatment has been shown to attenuate the inflammatory response associated with septicemia (septic shock) both locally and systemically and reduces associated liver and lung damage. 

Marker Gene has now developed a new long-wavelength fluorescent substrate Resorufin 7-O-Phosphate, di-ammonium salt (M1207) for use in these important phosphatase analyses, as well as the new MarkerGene^TM ResPhos^TM Alkaline Phosphatase Assay Kit (M1211) that includes this new long-wavelength phosphatase substrate and can be used to quantify phosphatase activity in solutions, in cell extracts, in live cells as well as on solid surfaces (such as PVDF membranes). The kit contains the highly purified Res-Phos phosphatase substrate (Ex/Em=571/583nm upon dephosphorylation), Calibration standard, Reaction buffer, Stop buffer, a broad-based phosphatase inhibitor cocktail and an optimized ‘mix and read' assay protocol that is compatible with HTS liquid handling instruments.  This new reagent and kit complement our existing products for phosphatase analysis, including the substrates 3-Phenylumbelliferone 7-O-Phosphate, hemipyridinium salt (M0541) and Fluorescein di-Phosphate, tetraammonium salt (FDP, M1034).  For more information about these new products, please visit our website or see the references below for more information.

• Rider D.A., Young S.P., (2003) "Measuring the specific activity of the CD45 protein tyrosine phosphatase." J. Immunol. Methods 277: 127-134.
• Pastula C., Johnson I., Beechem J.M., Patton W.F. (2003) "Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.” Comb. Chem. High Throughput Screen. 6: 341-346.
• Koller, E.,  Wolfbeis, O. S. (1985) “Syntheses and spectral properties of longwave absorbing and fluorescing substrates for the direct and continuous kinetic assay of carboxylesterases, phosphatases, and sulfatases.”  Monatsh. Chem. 116(1): 65-75.
•    Plaia,T.W.,et al.” Analysis of Embryonic Stem Cell Pluripotency Using a Dual-Color Fluorescence-Based Protocol.International Society for Stem Cell Research 2nd Annual Meeting.(2004).
• Kotobuki N., Hirose M., Funaoka H., Ohgushi H., (2004) “Enhancement of in vitro osteoblastic potential after selective sorting of osteoblasts with high alkaline phosphatase activity from human osteoblast-like cells.” Cell Transplant. 13: 377-383.
• Boronkai A., Than N.G., Magenheim R., Bellyei S., Szigeti A ., Deres P ., Hargitai B ., Sumegi B ., Papp Z ., Rigo J Jr ., (2005) “Extremely high maternal alkaline phosphatase serum concentration with syncytiotrophoblastic origin.” J.Clin.Pathol. 58: 72-76.
• Sánchez de Medina F., Martínez-Augustin O., González R., Ballester I., Nieto A ., Gálvez J ., Zarzuelo A (2004) “Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease.” Biochem.Pharmacol. 68: 2317-2326.
• Papaldo, P., Di Cosimo S., Ferretti G., Vici P., Marolla P., Carlini P., Fabi A., Cognetti F.,  (2004) “Effect of filgrastim on serum lactate dehydrogenase and alkaline phosphatase values in early breast cancer patients. Cancer Invest 22: 650-653.

Krabbe' Disease Lysosomal Glycosidase Assays.

The lysosomal storage diseases are a family of genetic human metabolic diseases that, in their severest forms, cause mortality as a result of progressive neurodegeneration. They are caused by mutations in the genes encoding glycohydrolase enzymes that catabolize glycosphingolipids within the lysosome.  When there is such a lysosomal enzyme deficiency, the deficient enzyme's undegraded substrates gradually accumulate within the lysosomes causing a progressive increase in the size and number of these organelles within the cell. This accumulation within the cell eventually leads to malfunction of the organ and to the gross pathology of a lysosomal storage disease, with the particular disease depending on the particular enzyme deficiency.  Krabbé disease is caused by a deficiency of galactocerebrosidase, an essential enzyme for myelin metabolism.   Deficiency of this enzyme causes accumulation of galactocerebrosides in various tissues.  Marker Gene has now developed a new, highly sensitive fluorescent substrate for measuring galactocerebrosidase activity inside of lysosomes, 2',7'-dichlorofluorescein di-galactoside (M1194).  This new substrate releases the highly fluorescent fluorophore 2',7'-dichlorofluorescein (EX: 495nm / EM: 529 nm) at the site of galactocerebrosidase activity, and since the pKa of the fluorophore is significantly lower, it retains appreciably more fluorescence in the highly acidic environment of the lysosome than other similar fluorophores.  Marker Gene also provides several other substrates for measuring levels of galactocerebrosidases including 4-Methylumbellifery-β-D-Galactopyranside (MUG, M0241), 4-Trifluoromethylumbelliferyl-β-D-Galactopyranoside (TFMU-Gal, M0252), Fluorescein di-β-D-Galactopyranoside (FDG, M0250) and Resorufin-β-D-Galactopyranoside (Res-Gal, M0203).  For more information about these assays and methods for measuring the benefits of individual substrates, please see the references below, or visit our website.

• van Es H.H., Veldwijk M., Havenga M., Valerio D. (1997) "A flow cytometric assay for lysosomal glucocerebrosidase" Anal. Biochem. 247: 268-271.
• Chan KW., Waire J., Simons B., (2004) "Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay." Anal. Biochem.  334(2): 227-33.
• Rudensky B., Paz E., Altarescu G., (2003)  "Fluorescent flow cytometric assay: a new diagnostic tool for measuring beta-glucocerebrosidase activity in Gaucher disease." Blood Cells Mol. Dis.  30(1): 97-9.
• Daniels L.B., Glew R.H., Diven W.F., Lee R.E., Radin N.S., (1981) “An improved fluorimetric leukocyte b-glucosidase assay for Gaucher's disease.” Clin. Chim. Acta 115: 369-375.
• Kaxpova E.A., Voznyi Ya V., Dudukina T.V., Tsvetkova I.V., (1991) “4-Trifluoromethylumbelliferyl glycosides as new substrates for revealing diseases connected with hereditary deficiency of lysosome glycosidases.” Biochem. Int.  24: 1135-1144.

Glycoprotein Analyses with a new Long-Wavelength Probe: M1202.

Fluorescent labeling of carbohydrates and glycoproteins has been a subject of significant research for many years.  Typical methods include reductive amination with aminonaphthalene trisulfonic acid (ANTS) or ethylenediaminonaphthalene sulfonic acid (EDANS).  Fluorescently labeled lectins have also been used to monitor sugars and glycoproteins on cell surfaces or in situ on gels or microplates.  A simpler and milder method of labeling sugars or oligosaccharides involves the use of phenylboronic acid derivatives such as fluorescein aminophenylboronic acid (EX 488nm / EM 519nm) or Dansylaminophenylboronic acid (M0329) (EX 337nm, EM 517nm).  These reagents provide a convenient method of analysis since they bind directly and reversibly to cis-diols (on sugars) at high pH values, either on cell surfaces and in vitro.  They can be used for carbohydrate determination of sugars or glycoproteins in serum, media, cell lysate or purified carbohydrate or glycoproteins samples as well as for HPLC analysis of released sugars or oligosaccharides from these sources.  Marker Gene has now developed a new long-wavelength fluorescent boronic acid derivative, Eosin 5-Thiouredylphenylboronic acid, triethylammonium salt (M1202) that can be used for these analyses, in conjunction with other fluorophores, for multiplexed analysis, in fluorescence energy transfer (FRET) studies or in a high-throughput format (EX 520/ EM544).  Eosin derivatives can also exhibit phosphorescence with an emission maximum at ~680 nm.  For more information about these new probes or assays for glycoproteins please see the references below, or visit our website. 

• Luis G.P., (1998) “Selective Fluorescent Chemosensor for Fructose." Analyst 123: 155.  
• Gamoh K., "Determination of Traces of Natural Brassinosteroids as Dansylaminophenylboronates by Liquid Chromatography with Fluorometric Detection." Anal. Chim. Acta 228: 101 (1990).
• Hayashi Y, Makino M., (1985)"Fluorometric Measurement of Glycosylated Albumin in Human Serum." Clin. Chim. Acta 149: 13-19.
• Vainio P., (1983) "N-(5-Dimethylaminonaphthalene-1-Sulfonyl)-3-Aminobenzene Boronic Acid as an Active-Site-Directed Fluorescent Probe of Lipoprotein Lipase." Biochim. Biophys. Acta 746: 217-219.
• Burnett T.J., Peebles H.C., Hageman, J.H., (1980) "Synthesis of a Fluorescent Boronic Acid Which Reversibly Binds to Cell Walls and a Diboronic Acid Which Agglutinates Erythrocytes." Biochem. Biophys. Res. Commun. 96: 157-162.
• Anthony P.F. Turner, Beining Chen ^a and Sergey A. Piletsky (1999) “In Vitro Diagnostics in Diabetes: Meeting the Challenge” Clinical Chemistry. 45: 1596-1601.
• Boeseken, J., (1949) Adv. Carbo. Chem. 4: 189-210.
• Solms, J., Deuel, H., (1957) Chimica 11: 311.
  
  

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.