Marker Gene Monthly Newsletter   

February, 2006

Volume 6, Number 2

© 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.

Thymidine Kinase as an in vivo Marker Gene.

Researchers at the Crump Institute for Molecular Imaging, the UCLA/DOE Laboratory of Structural Biology and Molecular Medicine and the UCLA School of Medicine, have developed new uses for the herpes simplex virus type I thymidine kinase (HSV1-tk ) gene as a reporter gene.  HSV1-TK has been widely used in gene therapy protocols for targeted drug delivery since it can phosphorylate the acycloguanosines acyclovir, ganciclovir and penciclovir, as well as thymidine analogs such as 2-fluoro-2-deoxy- D-arabinofuranosyl-5-iodouracil (FIAU).  Upon phosphorylation, these drugs become activated inside the transfected tissues, and are also “trapped” intracellularly due to their increased charge.  Positron-emitting analogs of these HSV1-tk substrates were synthesized, and modified HSV1-TK enzymes developed that permitted in vivo monitoring of HSV1-tk levels in transfected tissues by positron emission tomography (PET) through measurement of the enzymatically phosphorylated products.  This PET technique uses tracers of high specific activity and very low amounts of material such that the concentrations of the substrate are far below those used for pharmacologic killing of cells with this enzyme.  Among the drug analogs synthesized and evaluated were 8-[18F]fluoropenciclovir (FPCV), 9-[(4-[18F]fluoro-3-hydroxymethylbutyl)guanine, 3’-deoxy-3’-[18F]-fluorothymidine (FLT) and 8-[18F]fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene in cell culture and in vivo.  For more information about these new techniques for in vivo marker gene detection, please visit our website or see the references below.

• Yaghoubi, S.S., Wu, L., Liang, Q., Toyokuni, T., Barrio, J.R., Namavari, M., Satyamurthy, N., Phelps, M.E., Herschman, H.R., and Gambhir, S.S. (2001) Direct Correlation Between Positron Emission Tomographic Images of Two Reporter Genes Delivered by Two Distinct Adenoviral Vectors.  Gene Therapy 8: 1072-1080.
• Liang, Q., Satyamurthy, N., Barrio, J.R., Toyokuni, T., Phelps, M.P., Gambhir, S.S., and Herschman, H.R. (2001) Noninvasive, quantitative imaging, in living animals, of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction.  Gene Therapy 8: 1490-1498.
• Sun, X., Annala, A.J., Yaghoubi, S., Barrio, J.R., Nguyen, K., Toyokuni, T., Satyamurthy, N., Namavari, M., Phelps, M.E., Herschman, H.R., and Gambhir, S.S. (2001) Quantitative imaging of gene induction in living animals.  Gene Therapy 8: 1572-1579.
• Iyer M ,  Barrio J.R., Namavari M.,  Bauer E.,  Satyamurthy N.,  Nguyen K.,  Toyokuni T.,  Phelps M.E.,  Herschman H.R.,  Gambhir S.S., (2001) “8-[18F]Fluoropenciclovir: an improved reporter probe for imaging HSV1-tk reporter gene expression in vivo using PET.” J. Nucl. Med. 42(1): 96-105.
  
  

Artificial Chromosome Expression (ACE) Systems.

Artificial chromosomes represent a new and potentially promising approach for non-viral transformation and gene therapy applications.  Artificial Chromosome Expression (ACE) is an non-integrating expression system that can behave like a fully functional chromosome in a variety of mammalian cells (mouse, rat, rabbit, bovine and human).  It is made up primarily of satellite sequences found in pericentric heterochromatic DNA of mammalian chromosomes.  These sequences lack functional genes and when incorporated, seem to have no phenotypic effect on a host cell. The ACE is typically an engineered platform chromosome of up to 60 megabase pairs containing multiple site-specific integration sites.  This non-viral vector has the advantage that it does not interfere with other genomic sequences because it replicates as an episome in the target cell and it is mitotically stable in the absence of selection.  Unlike some viral vectors, the ACE does not integrate into the host genome, limiting concerns related to insertional modifications of host genes.   In addition, it has an almost unlimited cloning capacity that can allow for the insertion of a gene of interest, as well as other sequences required for correct or cell-specific expression.  Prototype ACE systems encoding approximately six copies of lacZ and eight copies of hygromycin-B resistance genes have been prepared, and more recently, an ACE carrying the gene for the red fluorescent protein has also been engineered.  But for these artificial chromosomes, which can be up to 1–2 microns in size, to be useful, efficient delivery to cells of interest must be achieved.  Commercially available cationic lipids are being investigated for use with ACE systems for transfection of living cells.  For more information about these new transfection vectors and systems please see our website or the references below.

• Telenius H, Szeles A, Kereso J, (1999) “Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.” Chromosome Res; 7: 3–7.
• deJong G, Telenius AH, Telenius H, (1999) “ Mammalian artificial chromosome pilot production facility: large-scale isolation of functional satellite DNA-based artificial chromosomes.” Cytometry; 35: 129–133.
• Hadlaczky G. (2001) “Satellite DNA-based artificial chromosomes for use in gene therapy.” Curr Opin Mol Ther; 3: 125–132.
• Lindenbaum M, Perkins E, Csonka E., (2004) “A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy.” Nucleic Acids Res; 32: e172.
• de Jong G, Telenius A, Vanderbyl S, (2001) “Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers.” Chromosome Res; 9: 475–485.
• Lipps HJ, Jenke ACW, Nehlsen K, (2003) “Chromosome-based vectors for gene therapy.” Gene; 304: 23–33.
• Vos JMH. (1997) “The simplicity of complex MACs.” Nat Biotechnol; 15: 1257–1259.
• Huxley C. (1997) “Mammalian artificial chromosomes and chromosome transgenics.” Trends Genet; 13: 345–347.
• Stewart S, MacDonald N, Perkins E, (2002) “Retrofitting of a satellite repeat DNA-based murine artificial chromosome (ACes) to contain loxP recombination sites.” Gene Ther; 9: 719–723.
• Ebersole, T. A., Ross, A., Clark, E., McGill, N., Schindelhauer, D., Cooke, H., & Grimes, B. (2000) "Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats", Hum.Mol.Genet., 9(11): 1623-1631.
• Masumoto, H., Ikeno, M., Nakano, M., Okazaki, T., Grimes, B., Cooke, H., & Suzuki, N. (1998) "Assay of centromere function using a human artificial chromosome", Chromosoma, 107(6-7):  pp. 406-416.
• Grimes, B. & Cooke, H., (1998) "Engineering mammalian chromosomes", Hum.Mol.Genet., 7(10): 1635-1640.
• Ikeno, M., Grimes, B., Okazaki, T., Nakano, M., Saitoh, K., Hoshino, H., McGill, N. I., Cooke, H., & Masumoto, H. (1998) "Construction of YAC-based mammalian artificial chromosomes", Nat.Biotechnol., 16(5): 431-439.

4-Methylumbelliferyl Galactoside (MUG).

The gene for lacZ ß-galactosidase is widely used for reporter gene studies. The recombinant ß-galactosidase enzyme from E. coli can be routinely manipulated and assayed to study promoter function, developmental regulation, tissue specific expression, mRNA stability, or signal sequences that target proteins to various organelles in vitro and in vivo.  lacZ ß-galactosidase has a wide substrate specificity and many substrates are available to report the activity of promoters and genes co-expressed with this marker gene.  b-Galactosidase activity can easily be revealed by using the fluorogenic substrate 4-methylumbelliferyl-b-D-galactopyranoside (M0241) in a sensitive and quantitative assay.  In assays, esters of 4-methylumbelliferone (4-MU) do not fluoresce unless cleaved by the enzyme to release the fluorophore.  Fluorometric enzyme assays based on the hydrolysis of 4-MU-containing substrates can be used for analysis of ß-glucuronidase (GUS) by 4-MU-glucuronide (M0240) or ß-galactosidase (lacZ) using 4-MU-galactoside (M0241).  Cleavage of 4-methylumbelliferyl-ß-D-galactoside by ß-galactosidase enzyme yields the fluorescent molecule 4-MU that emits light at 460 nm when excited by 365 nm light. In order to measure 4-MU for reporter gene assays, the ß-Gal producing cells are typically lysed and incubated with the appropriate substrate. It is important to note also that the recombinant E. coli lacZ ß-galactosidase activity is active at neutral pH, but the vertebrate form of ß-galactosidase is a lysosomal enzyme, which has optimal activity at pH 4.5.  Recombinant, versus cloned activities can therefore be independently measured using different pH systems for analysis.  To measure activity, cells are lysed, incubated with 4-MU containing substrate (1.0 mM concentration) and incubated for the same period of time, generally 10-40 minutes.  After reaction, 100 µL of the cell lysis incubation solution is added to 1.9 mL of 0.2 mM Carbonate Stop buffer to stop ß-Gal enzyme activity and raise the pH of the sample for more sensitive measurement.  Free 4-MU can be used as a standard to calibrate b-galactosidase activity in cell cultures or tissues.  A standard curve can be generated to examine the linearity of the assay within a particular concentration range, and for quantitating the enzymatic turnover rate.  It is recommended that you perform this at least once before performing the assay for the first time.  For more information about these assays and protocols, please visit our website or see the references listed below for more information.

• "A microplate fluorimetric assay for transfection of the beta-galactosidase reporter gene." Rakhmanova VA, MacDonald RC. Anal. Biochem. 257: 234-237 (1998).
• "Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A." Pshezhetsky AV, Elsliger MA, Vinogradova MV, Potier M. Biochemistry 34: 2431-2440 (1995).
• "Highly sensitive fluorimetric enzyme immunoassay for prostaglandin H synthase solubilized from cultured cells." Ruan KH, Kulmacz RJ, Wilson A, Wu KK. J Immunol. Methods 162: 23-30 (1993).
• "Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme." Young DC, Kingsley SD, Ryan KA, Dutko FJ. Anal. Biochem. 215: 24-30 (1993).
• "Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons." Kaang BK, Kandel ER, Grant SG. Neuron 10: 427-435 (1993).
• "Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer." Price J, Turner D, Cepko C. Proc. Natl. Acad. Sci. USA 84: 156-160 (1987).
• Ziebold, T. O. (1967) “Precision and sensitivity in electron microprobe analysis.” Anal. Chem. 39: 858.

Intron splicing for improved expression of GUS.

Agrobacterium tumefaciens is a commonly used vehicle for transforming dicot plants. The underlying mechanisms of transformation however are still not very well understood.  Researchers at the Institute for Gene Biology in Berlin recently used a GUS gene containing a portable intron (IV2 from the ST-LS1 gene) that could only be removed by eukaryotic splicing apparatus, not present in the agrobacterium (a prokaryote).  Upon transformation, the levels of GUS activity were monitored and indicated splicing from gene transfer and utilization throughout the whole cotyledon within 36 hours.  These data indicated efficient gene transfer and splicing in Arabidopsis with a GUS containing vector system.   pIG121 or pIG221 or pCAMBIA 1201, 1301, 2301 series vectors can be used to obtain the intron GUS.  It has also been reported that introns can enhance of gene expression in other species. (see also our WebNewsletter Jan. 2003). Methods used to detect GUS activity in plants and plant extracts include 4-Methylumbelliferyl-glucuronide (M0240), Carboxyumbelliferyl-glucuronide (M0256), and our new b-Glucuronidase (GUS) Reporter Gene Activity Detection Kit (M0877).  Please visit our website or see the references below for more information.

• Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M., (1990) “Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.”  Mol. Gen. Genet. 220(2):245-50.
• Song GQ; Sink KC (2004) “Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).”  Plant Cell Rep. 23(7): 475-84
• Song G.Q., Sink K.C. (2006) Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.  Plant Cell Rep. 25(2): 117-23.
  
  

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.