Marker Gene Monthly Newsletter   

January, 2004

Volume 4, Number 1

© 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.

Gene Silencing Vectors containing Marker Genes.

Doubly-stranded RNA (dsRNA), when introduced into cells containing the same sequence, initiate degradation of the equivalent messenger RNA and depletion of the protein in vivo.  Such posttranscriptional gene silencing (PTGS) may be a means of protecting cells from transposons or viruses that can produce dsRNA.  Within the cell, the dsRNAs are enzymatically cut into smaller RNA pieces, which then bind to homologous mRNA. RNA fragments longer than about 30 nucleotides, however, destroy cellular mRNA, resulting in cytotoxicity and apoptosis.  Therefore small interfering RNA (siRNA) fragments of about 21-23 nucleotides long are the most common size used in regulation systems.  The two most common methods of integrating these silencing siRNA’s into cells involve either transfection by a plasmid or viral vector or by direct introduction of chemically synthesized siRNA into cells.  Expression vectors have the advantage of longer persistence of the knockdown effect and the potential for genetic rescue.  These vectors typically contain a small hairpin sequence that induces self-complementation and double stranding.  The nuclease DICER, removes the hairpin, releasing the final siRNA.  Several new vectors have been developed that contain these elements as well as the lacZ and GFP marker genes for use in FACS cell sorting, single-cell analysis (photomicroscopy) and gene insertion studies.  For more information about these exciting new technologies, see the references below:

• Hammond S.M., Caudy A.A., Hannon GJ. (2001) “Post-transcriptional Gene Silencing by Double-stranded RNA”. Nature Rev Gen 2: 110-119.
• X.F. Qin, D.S. An, I. S. Y. Chen  D. Baltimore (2003) “Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5” PNAS 100(1): 183-188.
• Xia H, Mao Q, Paulson HL, and Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotech 20: 1006-1010.
• S. Kojima, D. Vignjevic, and G. G. Borisy (2004) “Improved silencing vector co-expressing GFP and small hairpin RNA” Biotechniques 36:74-79.
• Y. Katagiri K.C. Ingham (2002) “Enhanced Production of Green Fluorescent Fusion Proteins in a Baculovirus Expression System by Addition of Secretion Signal” BioTechniques 33:24-26.
• NOTE: An siRNA search engine has recently been developed by Bingbing Yuan and Fran Lewitter of the Whitehead Institute for Biomedical Research. Its use is free of charge for academic users. The program output is ranked by the degree of specificity of the predicted siRNAs. The user is able to define sequence search patterns and can exclude single-nucleotide polymorphic sites from siRNA predictions.  Click the link to access this site.

Senescence Induction Analysis.

Many primary cell types, particularly fibroblast cells, have a limited capacity to reproduce in cell culture.  Even normal cells derived from fetal, embryonic or newborn tissue, typically undergo between 40 and 60 cell divisions, but then often stop dividing.  This number is often referred to as the Hayflick Limit.  The change to a non-dividing state is accompanied by changes in morphology, shape, and gene expression patterns.  In addition, senescence also is accompanied by increases in intracellular and secretory enzymatic activity.  Recent studies have shown that b-galactosidase histochemical staining at pH 6 is a useful enzymatic marker of cellular senescence (SA-b-Gal).  While cells entering senescence show this enzymatic activity, immortalized cell types, including tumor or other quiescent cells, are not stained under these conditions.  The implications of these methods in such diverse areas as age-related pathology research, tumor analysis and tissue culture maintenance is significant.  Marker Gene provides several substrates for intracellular analysis of b-galactosidase activity including FDG (M0250), Res-Gal (M0203) and CMUG (M0257) which can also differentiate pH at the site of enzyme action.  For more information about these new assays, see the references below, or visit our Website:

• GP Dimri, X Lee, G Basile, M Acosta, G Scott, C Roskelley, EE Medrano, M Linskens, I Rubelj, O Pereira-Smith, M Peacocke, and J Campisi (1995) ”A Biomarker that Identifies Senescent Human Cells in Culture and in Aging Skin in vivo”
  PNAS 92: 9363-9367.
• Nakamura M., Kondo H., Shimada Y., Waheed A.A., Ohno-Iwashita Y.,  (2003) “Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts.” Exp. Cell Res. 290(2): 381-90.
• Aoshiba K; Tsuji T; Nagai A (2003) “Bleomycin induces cellular senescence in alveolar epithelial cells.” Eur. Respiratory J. 22(3): 436-43.

Removal of Marker Genes by Recombination.

Marker genes find numerous applications in animal and plant systems, but can also cause consumer concern when used in commercial products or add regulatory requirements from the presence of "excess" exogenous DNA.  Several methods have recently been introduced to remove marker genes in plants, using site-specific recombination systems (e.g., lox/Cre recombinase, Flp recombination target (FRT)/Flp recombinase, or Rs/R recombinase) and by using an inducible promoter system (e.g. for b-estradiol) for the recombinase gene.  In these recombination schemes, the marker gene is flanked by recombination sites, such as lox, FRT, or Rs, which specifically interact with a recombinase protein (e.g., Cre, Flp, or R, respectively).  This interaction promotes recombination between the sites and deletes the marker DNA from the host genome.  The Cre-lox technology involves site-specific recombination of DNA using Cre recombinase and lox DNA sites. Its usefulness in basic medical research is widely recognized. Cre-lox site-directed recombination has shown great utility in developing transgenic mouse models where specific genes can be deleted at specific times and in specific tissues. Marker Gene is working with prominent researchers; including Dr. David Ow at the Plant Gene Expression Center to develop kits for use of these systems by the research community.  For more information about these techniques please see the references below or visit our Website.

• David W. Ow “The right chemistry for Marker Gene removal?” (2001) Nature Biotechnol. 19(2): 115 – 116.
• Zuo, J., Niu, Q.-W., Moller, S.G. & Chua, N.-H. “Chemical-regulated, site-specific DNA excision in transgenic plants.” Nat. Biotechnol. 19, 157-161 (2001).
• Sugita, K., Kasahara, T., Matsunaga, E. & Ebinuma. H.” A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency” Plant J. 22, 461-469 (2000). 
• Sauer, B. “Site-specific recombination of DNA in eukaryotic cells”, U.S. Patent 4,959,317, Sept. 25, 1990.

Transgenic LacZ mouse models.

The use of reporter genes in vivo can allow examination of spatial patterns of gene expression of a particular promoter within a tissue, embryo, or adult mouse.   The E. coli lacZ gene has been widely used in this context, and when integrated into a mouse genome by transgenic techniques, can be used as a reporter gene under the control of a given promoter/enhancer in a transgene expression cassette.   Transgenic animals have been used to identify factors and conditions that modulate the expression profile of the specific promoter or enhancer.  The Jackson Laboratory has several pf these mouse strains available for such studies.  These GEMM^TR strains were constructed either by retroviral transfection or by incorporating lacZ into embryonic stem (ES) cells by homologous recombination. The genetically-modified ES cells were then microinjected into host embryos at the 8-cell blastocyst stage.  Microinjected embryos were transferred into pseudopregnant host females that bore chimeric progeny. The chimeric progeny that carry the lacZ gene in their germ line were then bred to establish the line.  See the Jackson Laboratory web site or the references below for more information on these exciting research models.

• Redfern CH, Coward P, Degtyarev MY, Lee EK, Kwa AT, Hennighausen L, Bujard H, Fishman GI, Conklin BR. (1999) “Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice”. Nat Biotechnol 17:165-9.
• Schmidt A, Tief K, Foletti A, Hunziker A, Penna D, Hummler E, Beermann F. (1998) “lacZ transgenic mice to monitor gene expression in embryo and adult.” Brain Res Brain Res Protoc 3(1): 54-60.
• Monastersky GM, Robl JM, eds. (1995) "Strategies in Transgenic Animal Science." American Society for Microbiology Press. Washington, DC.
• Mercer EH, Hoyle GW, Kapur RP,Brinster RL, Palmiter RD. (1991) The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice. Neuron 7: 703-716.
• Boerrigter ME, Dolle ME, Martus HJ, Gossen JA, Vijg J. (1995) “Plasmid-based transgenic mouse model for studying in vivo mutations”. Nature 377: 657-9.
  
  

Firefly Luciferase Assays in Tissues.

Analysis of cloned Luciferase (luc) activity in live mammalian cells can be accomplished using a variety of methods, including lysis assays and microtiterplate systems, but it is often useful to recover the cells under analysis for later expansion or secondary analysis.  Our Luciferase Assay Kit (M0626) contains the reagents, buffers and a detailed protocol for live cell luc analysis in culture, by microscopic analysis, microplate analysis or using the common lysis assay conditions.  In addition, analysis of tissue samples can now be accomplished using our updated protocol.   The Live Cell Luciferase Assay Kit contains sufficient reagents for up to 1000 assays, using the conditions described.  For more information about these assays and other reagents useful for luciferase detection, see the references below or visit our Web site.

• Maechler, P., Wang, H., Wollheim, C.B. “Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase.” (1998) FEBS Lett. 422: 328-332.
• Takasuka, N., White, M.R.H., Wood, C.D., Robertson, W.R., Davis, J.R.E., “Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells.” (1998) Endocrinology 139(3): 1361-1368.
•  J.J. Gildea, et al. (2000) "Transmembrane Motility Assay of Transiently Transfected Cells by Fluorescent Cell Counting and Luciferase Measurement." Biotechniques 29: 81.
  
  

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.
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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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