Marker Gene Monthly Newsletter   

April, 2003

Volume 3, 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.

Combined lacZ and GFP Detection.

Green Fluorescent Protein (GFP) and lacZ b-Galactosidase are both popular reporters of gene expression.  Unfortunately the emission properties of GFP overlap with the most common and sensitive lacZ detection system (FACS-Gal/FDG).  Recently, a combined GFP and lacZ staining protocol using the red fluorescent b-Gal substrate C-12Res-Gal (dodecylresorufin b-D-Galactopyranoside) has been developed.  In this method, the two gene expression events are easily detected independently.  GFP fluorescence was detected using an excitation filter at 310 nm and an emission filter at 510 nm, while resorufin was detected by using an excitation filter at 520 nm and an emission filter at 620 nm.  Marker Gene also sells a new FACS lacZ ß-Galactosidase Detection Kit (Product M0255) which produces a blue fluorescent product upon lacZ b-galactosidase activity.  Please see the references below for more information about these techniques:

• Zhang Y, Naleway J, Larison K, Huang Z, Haugland R. (1991) Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic B-galactosidase substrates. FASEB 5:3108-3113.
• Witrup K, Bailey, J. (1988) “A Single-Cell Assay of b-Galactosidase Activity in Saccharomyces cerevisiae. Cytometry. 9:394-404.
• https://www.marbec.org/education/projects/2000/report_gabler.pdf
  

ß-Lactamase as a Marker for Gene Expression. 

Expression of ampicillinase activity (amp, ß-lactamase) is a common marker gene used for selection of recombinant cells in culture.  Several colorimetric ß-lactamase substrates, including nitrocefin, cephacetrile and PADAC have been described.  Recently, a membrane permeable fluorogenic substrate for ß-lactamase (CCF2/AM) has been developed which allows the detection of enzyme activity in intact mammalian tissue culture cells.  The substrate consists of two fluorophores (6-chloro-7-hydroxycoumarin and fluorescein) attached to a cephalosporin ring bringing them into close proximity, allowing for efficient fluorescence resonance energy transfer (FRET).  When the substrate is excited at 409nm, emission of 520 nm (green) is observed.  b-Lactamase activity, however, releases the fluorecein, resulting in disruption of the FRET and a shift to the emission to 447 nm (blue).  For more information about these techniques for amp detection, please see the references below.

• E. Raz,  G. Zlokarnik, R.Y. Tsien, W. Driever, (1998) “b-Lactamase as a Marker for Gene Expression in Live Zebrafish Embryos” Dev. Bio 203: 290–294.
• Zlokarnik, G., Negulescu, P., Knapp, T., Mere, L., Burres, N., Feng, L., et al. (1998). Quantitation of transcription and clonal selec-tion of single living cells with beta-lactamase as reporter. Science 279, 84–88.
• Jones RN, Wilson HW, Novick WJ Jr. (1982) “In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds.” J Clin. Microbiol. 15(4):677-83.

Fungal Identification using PCR Analysis.

Historically, the phylogeny of fungi has been inferred from various phenotypic characterizations, such as morphology, physiology or development.  PCR analysis of gene expression has advanced specific detection and identification of existing and new fungal species and will continue to be useful for control of parasitic and rare taxa.  Marker Gene recently introduced a new PCR based Citrus Black Spot Assay (M0796) for identification of the pathogenic Guignardia citricarpa strain   Please see the following references for more information about PCR analysis of fungal strains, or visit our Web site for additional information and our new PCR-based assays under development.

• Frederick, R.D. Snyder, K.E., Tooley, P.E., Berthier-Schaad, Y., Peterson, G.B., Bonde, M.R., Schaad, N.W., and Knorr,, D.A. 2000. Identification and differentiation of Tilletia indica and T. walkeri using the polymerase chain reaction. Phytopathology 90: 951–960.
• James Borneman, R. Jack Hartin (2000) “PCR Primers That Amplify Fungal rRNA Genes from Environmental Samples” Appl. Environ. Microbiol.  66 (10): 4356–4360.

Secondary Screening of Blue-White Colonies.

Blue-white screening is one of the most popular methods of screening bacterial colonies for recombinant plasmid insertion.  Upon gene insertion into the lacZ gene site, the plasmids lose ß-galactosidase activity, and when stained on X-Gal containing agar plates, appear as white colonies.  Blue colonies will contain plasmids without the insert.  Picking white colonies for amplification, however, is often inaccurate, and can lead to up to 40% of false-positives for the insertion.  Secondary screening of the colonies in LB media containing various concentrations of X-Gal, ampicillin and/or inducer IPTG, can be used to further select positive insert clones.  Overnight growth in a shaker bath will reveal false-positives as blue or green, while true-positives remain clear (see figure at left).  Please see the references below for more information about these techniques.

•  Sherwood, A.L., “Virtual Elimination of False Positives in Blue-White Colony Screening” Biotechniques 34:644-647. 
• http://www.inbios.com/readytogrow.html
  

New Catalog Now Available!

The 2003-2004 edition of the Marker Gene catalog is now available on-line on our  Web site.  Many new products and kits, additional literature references, data and protocols have been included, as well as new information about our old products.  If you would like a print copy delivered directly to your office or lab, 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.
Check it out 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. 

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