Marker Gene Monthly Newsletter   

October, 2003

Volume 3, Number 10

© 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.

FLP and Cre Recombinases Induce GFP and lacZ.

Site-specific recombinases (SSRs) are enzymes from bacterial and yeast sources that are able to cut and then ligate DNA at specific targets inducing recombination. The most common SSRs are two members of the integrase family, Cre recombinase from the bacteriophage P1, which recognizes a 34 bp sequence called loxP, and FLP recombinase from Saccharomyces cerevisiae, which recognizes the 34 bp FRT site. Cre and FLP recombinases carry all functions required for the recombination event in single polypeptide chains.  Consequently, both Cre and FLP have been used for genome engineering in bacteria, yeasts, plants, flies, mammalian cells and mice.  By incorporating the recombination sites between a promoter element and a gene of interest (GFP or lacZ marker genes, for example), recombination can be used to track, control and induce gene expression events or for conditional gene silencing.  For more information about these new systems, see the references below, or visit our web site and information on our products (M0250, M0259 and M0855).

• “Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites.”, Sternberg,N. and Hamilton,D. (1981) J. Mol. Biol., 150, 467-486.
• “Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast.”, Falco,S.C., Li,Y., Broach,J.R. and Botstein,D. (1982) Cell, 29, 573-584.
• “The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli.”, Cox,M.M. (1983) Proc. Natl. Acad. Sci. USA, 80, 4223-4227.
• “Functional expression of the Cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae”, Sauer,B. (1987) Mol. Cell. Biol., 7, 2087-2096.
• “Site-directed recombination in the genome of transgenic tobacco.” Odell,J., Caimi,P., Sauer,B. and Russell,S. (1990) Mol. Gen. Genet., 223, 369-378.
• “Tissue- and site-specific DNA recombination in transgenic mice.”, Orban,P.C., Chui,D. and Marth,J.D. (1992) Proc. Natl. Acad. Sci. USA, 89, 6861-6865.
• "Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination." Sabath DE, Shim MH. Biotechniques 28: 966-972 (2000).
  

The Ice Nucleation Protein (InaZ) Reporter Gene.

Contrary to the popular misconception, water does not always freeze at 0^oC, but can remain liquid when supercooled to nearly  –40^oC, if free of ice nucleating species.  Supercooling to temperatures lower than –15^oC rarely occurs in nature due to the widespread presence of ice nuclei.  Almost all organic and inorganic substances can catalyze ice formation (i.e. serve as ice nuclei) at temperatures between –15^oC and –40^oC, and certain compounds can serve as nuclei at temperatures as high as –6^oC.  But in the absence of biological ice nuclei, water will typically supercool at temperatures of –6^oC or lower without freezing.   This property has been used to develop a new marker gene, for use in microbiological and plant sciences.  The ice nucleation activity of P. syringae has been found to be conferred by a single gene (inaZ) that encodes an outer membrane protein (InaZ).  Individual InaZ  proteins cannot serve as ice nuclei, but form large, homogeneous aggregates that collectively orient water molecules into a conformation mimicking the crystalline structure of ice, thereby catalyzing ice formation.  Oriented water molecules freeze at temperatures slightly below zero (i.e. –2^oC to -10^oC) instead of supercooling, so assays of individual cells (microtiterplate format) cooled to precisely  –6^oC can be used to identify cells that are transgenic.  For more information about this new marker gene, see the references below:

• William G. Miller, Maria T. Brandl, Beatriz Quiñones, and Steven E. Lindow^,, “Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes.” Applied and Environmental Microbiology (2001) 67(3):1308-1317.
• Joyce E. Loper and Steven E. Lindow. 1997. “Reporter gene systems useful in evaluating in situ gene expression by soil- and plant-associated bacteria.” p. 482-492. In Christon J. Hurst (ed. in chief), Manual of Environmental Microbiology. ASM Press, Washington, DC.
• Warren, G. J. 1995. “Identification and analysis of ina genes and proteins”, p. 85-99. In R. E. Lee, Jr., G. J. Warren and L. V. Gusta (ed.), Biological Ice Nucleation and Its Applications. APS Press, St. Paul, Minnesota.
• Vali, G. 1971. “Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids.” J. Atmos. Sci. 28:402-409.
• Maria Marco and Steve Lindow “Quantification Of Biological Ice Nuclei From The inaZ Reporter Gene: The Droplet Freezing Method”  http://plantbio.berkeley.edu/~bacteria/protocol%2017.pdf

Nuclear Compartmentalization Causes Down-regulation of Marker Genes.

Gene expression in mammalian cells may be mediated by positioning of a gene in proximity to the nuclear compartment in transfected cells.  In gene therapy applications, transgenes (lacZ) have been found localized in the nuclear periphery of down-regulated hosts within 36 hours after transfection, compared to expressing hosts.  Detection of marker genes and their encoded protein products using a combination of fluorescent probes indicate that nuclear compartmentalization may play a role in down-regulation of reporter transgenes by means of peripheralization, extrusion and/or degradation.  For more information on these techniques and improvements in transgene expression, see the references below:

•   “The contribution of nuclear compartmentalization to gene regulation.”, (2002)  Carmo-Fonseca M., Cell. 108(4): 513-21.
•   “Nuclear compartmentalization and gene activity during cellular differentiation.” (2002) C. Francastel, M. Groudine, Cancer Detection and Prevention V 26:295.  
• “Long-term expression driven by herpes simplex virus type-1 amplicons may fail due to eventual degradation or extrusion of introduced transgenes.” (2000) Tsai, D.J., Ho, J.J., Ozawa, C.R., Sapolsky, R.M., Exp. Neurol. 165(1): 58-65.

High-Efficiency Transfection using “Adenofection”.

Transient transfection of polarized epithelial cells or retinoblastoma cell types is difficult because of the tight cell connections and from the difficulties in producing recombinant virus with varying mutational or promoter fragments.  Recently a number of new techniques have been introduced which can improve transfection efficiency in these cell lines.  By noncovalently coupling an adenovirus particle to a plasmid DNA (polyethylenimine (2000 MW) or polylysine) transfection efficiencies were improved (3-10X over conventional lipofection agents), and the post-transfection expression levels were found to peak quicker (within 10-12 hours after application).   These “adenofection” systems typically use inactivated (psoralen-treated) adenovirus, which completely blocks gene expression from the virus genome.   For more information about these new techniques, see the references below:

• Bischof, J., Vietor, I., Cotton, M., Huber, L.A. “Transient Transfection of Mammary Epithelial Cells with a PEI/DNA/Adenovirus System”, (1999) Biol. Chem. 380:269-273.
• White, B.J., Taylor, R.E., Pittler, S.J., “Reproducible high efficiency gene transfer into Y79 retinobalstoma Cells using adenofection” (2001) 106:1-7.
  
  

New Renilla, Gaussia and Pleuromamma luciferases

Marine luciferases catalyze the oxidation of the small molecule coelenterazine, to produce light. Unlike the firefly luciferase systems, these coelenterazine-based luciferases do not require accessory high-energy molecules such as ATP for their signal, simplifying their use in a number of new reporter applications. The most common form is the luciferase isolated from the bioluminescent sea pansy Renilla reniformis. Gaussia luciferase, isolated from the copepod Gaussia princeps, when expressed in mammalian cells has been found to be up to 750-fold brighter than native Renilla reniformis luciferase.  For more information about these exciting new marker gene systems see the references below.  Look for new products from Marker Gene in these areas soon!

• “An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae.” Harger J.W., Dinman J.D., RNA ( 2003) 9(8): 1019-24.
• “Bioluminescent molecular imaging of endogenous and exogenous p53-mediated transcription in vitro and in vivo using an HCT116 human colon carcinoma xenograft model.”, Wang W.,El-Deiry W.S., Cancer Biol. Ther . ( 2003) 2(2): 196-202.
• “Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.”, Paulmurugan R., Gambhir S.S., Anal. Chem ., ( 2003) 75(7): 1584-9.
• “A new reporter gene for transient transfection of Plasmodium falciparum.” Militello K.T., Wirth D.F., Parasitol Res ( 2003) 89(2): 154-7.
• “Detecting protein-protein interactions using Renilla luciferase fusion proteins.”, Burbelo P.D., Kisailus A.E., Peck J.W., Biotechniques (2002) 33(5): 1044-8, 1050.
  
  

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.