Marker Gene Monthly Newsletter - Volume 12, Number 1 - January, 2012

Use of Melanoma Specific Promoters

Melanomas are aggressive tumors that continue to be among the most dangerous of skin cancers and against which there are few effective therapies. Targeting melanoma cells for gene therapy has recently been achieved using the human tyrosinase promoter (Tyr) which normally regulates production of the tyrosinase enzyme, a key enzyme in the production of melanin in the pigmented melanoma cells. The use of the Tyr promoter for GDEPT gene therapy in melanomas has been demonstrated by Dr. Brian Hughes and colleagues at the University of Alabama who have shown that transfection of melanoma cells with a purine nucleoside phosphorylase gene from E. coli under control of the Tyr promoter affects conversion of non-toxic 6-methylpurine-deoxyriboside into the highly toxic 6-methylpurine by the encoded enzyme in the transfected tumors.

Further study of the action of the Tyr promoter in melanoma cells has been undertaken by Drs. Martinelli and De Simone at the University of Naples, Italy who have synthetically engineered promoters using certain elements of the tyr promoter as building blocks. They were able to demonstrate about a 6-fold increase in activity using the CAT marker gene by these new promoter elements compared to the endogenous promoter. This type of targeted expression using tumor-specific promoters as well as other cis-elements to further target expression to only tumor cells holds the promise of new therapeutic advances in this area. For more information about these techniques, please see the references below or visit our website.

• Martinelli R, De Simone V. (2005) “Short and highly efficient synthetic promoters for melanoma-specific gene expression.” FEBS Lett. 579(1):153-156.
• Hughes BW, Wells AH, Bebok Z, Gadi VK, Garver RI Jr, Parker WB, Sorscher EJ. (1995) “Bystander killing of melanoma cells using the human tyrosinase promoter to express the Escherichia coli purine nucleoside phosphorylase gene.” Cancer Res. 55(15): 3339-3345.
• Bentley N, Eisen T, Goding CR. (1994) “Melanocytespecific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.” Mol. Cell. Biol. 14, 7996–8006.
• Vile RG, Russel SJ, Lemoine NR. (2000) “Cancer gene therapy: hard lessons and new courses.” Gene Therapy 7: 2–8.
• Nettelbeck DM, Rivera AA, Balagué C, Alemany R, Curiel DT.(2002) "Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter." Cancer Res. 62(16): 4663-4670.

Upconverting Nanophosphors for Bioimaging

High resolution fluorescence imaging using standard fluorophores typically requires excitation at short (UV) wavelengths which can cause autofluorescence and phototoxicity for cellular analyses. Quantum dots can have a much brighter emission but also contain heavy metals that can be cytotoxic. To help with these limitations, a series of new fluorescent nanoparticles called upconverting nanophosphors (UCNPs) has been developed. These particles consist of a host material such as yttrium oxysulfide co-doped with rare earth elements such as ytterbium, and have the ability to absorb lower energy long-wavelength incident light and emit at shorter (higher-energy) visible wavelengths often utilizing excitation in the near-IR region with emission at the visible wavelengths. Since this long wavelength excitation falls outside the typical excitation spectrum of cellular components, autofluorescence is minimized while the short wavelength emission still allows for detection with standard instrumentation. Additionally, these synthetic nanophosphors can be customized to emit at multiple wavelengths dependent on the excitation wavelength used.

Because these particles are often quite small (40-50 nm), incorporation into biological assays by attachment to proteins, antibodies or other tags has the promise to open up imaging possibilities way beyond that currently possible with current fluorophores. This has been demonstrated by Dr. Peter Choyke and colleagues at the U.S. National Cancer Institute where they applied the UCNPs to track the mouse lymphatic systems allowing less invasive surgery techniques to be utilized during cancer treatment. For more information about these techniques, please see the references below or visit our website

• Rosenblum LT, Kosaka N, Mitsunaga M, Choyke PL, Kobayashi H. (2010) “In vivo molecular imaging using nanomaterials: general in vivo characteristics of nano-sized reagents and applications for cancer diagnosis. Mol. Membr. Biol. 27(7):274-85.
• Lim SF, Riehn R, Tung CK, Ryu WS, Zhuo R, Dalland J, Austin RH. (2009) “Upconverting nanophosphors for bioimaging.” Nanotechnology. 20(40):405701.
• Ungun B, Prud'homme RK, Budijon SJ, Shan J, Lim SF, Ju Y, Austin R (2009) "Nanofabricated upconversion nanoparticles for photodynamic therapy," Opt. Express 17, 80-86.

Using Luciferase to Measure of DNA Methyltransferase Activity

DNA methylation is an important process in normal cell development and differentiation. The enzyme DNA methyltransferase (MTase) adds a methyl group to the 5-position of the pyrimidine ring of cytosine bases, thereby altering the expression of the affected genes. This process is integral for normal gene regulation and initial translation of a genome into an organism. Abnormal methylation has been shown to be involved in tumorigenesis, and the ability to measure the activity of the MTase enzyme is important to improve our understanding of cancer development.

Recently, a novel assay for measuring the activity of MTase has been described by  Dr. Cheng Jiang and colleagues at Hunan University, China using an ultrasensitive luciferase assay. This assay uses DNA encoding the luciferase gene and exploits the properties of some restriction enzymes that are sensitive to DNA methylation and therefore unable to cut in these regions. The assay functions by allowing the MTase to act on the DNA at varying concentrations (or with varying levels of inhibitors) and then digesting with the methylation sensitive enzymes and expressing the resulting protein in vitro. The resulting luciferase activity acts as a monitor of MTase activity, with the premise that methylated DNA will not be digested and go on to be translated into protein, whereas the unmethylated DNA will be cut by the restriction enzyme and not successfully code for luciferase. Thus the activity of luciferase is directly proportional to that of the MTase. This assay provides an sensitive test for MTase over a wide range of values that could provide a vital insight into the role of MTase and potential new inhibitors for cancer therapy or in developmental analyses. For more information about these techniques, please see the references below or visit our website  

• Jiang C, Yan CY, Huang C, Jiang JH, Yu RQ (2012) “A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage.” Analytical Biochemistry (available online Jan. 24, 2012).
• Daura-Oller E, Cabre M, Montero MA, Paternain JL, Romeu A (2009). "Specific gene hypomethylation and cancer: New insights into coding region feature trends". Bioinformation 3 (8): 340–343.
• Jaenisch, R.; Bird, A. (2003). "Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals". Nature genetics 33 Suppl (3s): 245–254.

Chimeric Primates Created in Oregon

Research into chimeric animals began with the creation of the first mouse chimeras from multiple embryos in the 1960s. These techniques, with some refinements, have proved invaluable in exploring the mechanisms of development, the methods by which embryos become differentiated and monitoring differing patterns of gene expression. The mouse chimeric model, while highly useful, has shown limitations when used to elucidate information as related to human development.

Recently the first generation of a primate chimera has been accomplished by Dr. Shoukhrat Mitalipov and colleagues at the Oregon Health & Science University. These chimeric primates have suggested that the mouse model does not give as good a reference for human development as once thought. Unlike mice studies, generation of primate chimeras was not possible by injection of embryonic stem cells or injection of whole inner cell mass into blastocysts. Only by the mechanical fusing of 3-6 embryos at the 4 cell stage could chimeric offspring be produced. The chimeric animals that resulted showed full chimerism throughout their organs and tissues. In addition, viable infants showed no evidence of any birth defects or enlargement as a result of being generated from multiple embryos. In the early stages of the experiments, the marker gene GFP was expressed in one set of cells and used to track the aggregation of cells from different embryos into a single embryo. This data was then utilized to create the final chimeric animals.

This work could have repercussions for the use of therapeutic stem cells as it showed that primate stem cells, cultured in vitro, do not show the same level of incorporation back into a host embryo as in other species. The current understanding of the mechanisms underlying these processes remain uncertain, however. For more information about these techniques, please see the references below or visit our website.

• Tachibana M, Sparman M, Ramsey C, Ma H, Lee HS, Penedo MCT, Mitalipov S (2012) “Generation of Chimeric Rhesus Monkeys” Cell, 148(1–2): 285-295.
• Tarlowski AK (1961)” Mouse chimaeras developed from fused eggs.” Nature 190: 857-860.
• Tucker EM, Moor RM, Rowson LE. (1974) “Tetraparental sheep chimaeras induced by blastomere transplantation. Changes in blood type with age.” Immunology 26(3): 613-621.
• Beddington RS, Robertson EJ. (1989) “An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo.” Development 105(4):733-737.

Compare Our Quality

Marker Gene strives to offer our customers products of the highest quality at competitive prices. Our years of experience allow us to provide excellent products in a timely manner. For more information, visit our website at http://www.markergene.com/ and click on the "PRODUCTS" link . We think you will appreciate our efforts to maintain excellent quality in our items for your research. For more information about any of our products, simply call 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 information about our products and their specifications.

CONTRACT RESEARCH @ markergene.com

• 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.

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.