Marker Gene Monthly Newsletter - Volume 10, Number 1 - January, 2010

Haitian Relief Fund Donation.

During the month of February, Marker Gene will donate 10% from the revenues of all orders placed online at www.markergene.com to the Save the Children’s Support the Haiti Earthquake Children in Emergency Fund. Thank you for your help in our efforts to help the people of Haiti during their time of need. If you would like more information about the fund, please click on the link above.

Red Shifted Renilla Luciferase.

One of the key elements precluding the use of marker genes in vivo stems from the inherent absorption of shorter wavelengths of light by endogenous compounds found in live tissues. Therefore, shifting emission toward longer wavelengths (above the so-called "therapeutic window" of approximately 650 - 1100 nm) where many tissues become partially transparent can allow for improved sensitivity and lowered detection limits in live animal studies. Native Renilla luciferase (RLuc) is a monomeric, bioluminescent protein derived from the sea pansy (Renilla reniformis), that codes for a 36 kDa enzyme catalyzing the oxidation of the substrate coelenterazine to coelenteramide, resulting in the production of visible light (at 481nm) that can be measured in live tissues. This RLuc emission at 481 nm has also been used in the presence of GFP (green fluorescent protein) in a bioluminescence resonance energy transfer (BRET) system to produce fluorescent light emission at 509 nm.

But recent work from the laboratories of Prof. S Sam Gambhir and co-workers at the Molecular Imaging Program at Stanford Univ. have developed a red-shifted RLuc variant (RLuc8.6-535). By using mutagenesis of amino acid residues near the enzyme active site and screening of these RLuc variants, they found the new RLuc8.6-535 luciferase variant to exhibit a three-fold increase in sensitivity at non-superficial tissue depths over the native protein with emission wavelengths shifted to 535 nm. The new enzyme also exhibited improved enzymatic activity (about 2-fold) compared to the native luciferase as well as enhanced stability in vivo. The implications of this new, more-sensitive luciferase for live animal monitoring of gene activity are significant. For more information about in vivo analysis with luciferase, please see our website or the references below.

• Loening AM, Dragulesscu-Andrasi A, Gambhir SS (2010) "A Red-shifted Renilla luciferase for transient reporter-gene expression." Nat. Meth. 7(1): 5-6.
• Koterba KL, Rowan BG (2006) "Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET)." Nuclear Receptor Signaling.4:e021. doi: 10.1621/nrs.04021
• Loening AM, Fenn TD, Wu AM, Gambhir SS (2006) "Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output." Protein Eng. Des. Sel. 19(9): 391-400.
• Loening AM, Wu AM, Gambhir SS (2007) "Red-shifted Renilla reniformis luciferase variants for imaging in living subjects." Nat. Meth. (8):641-643.

Bacterial Phenotype MicroArrays.

Evolutionary forces have driven bacteria to grow in as many environmental niches as possible. Species have formed by environmental selective pressures to produce different strains that are able to survive and grow in the multitude of niches in the environment. Various aspects of an environment, including toxic chemicals, temperature, pressure, light, desiccation, nutrients and others have produced what has been estimated to be up to 10⁹ different bacterial species, of whom only about 9000 have thusfar been described and named (see LSPN). Researchers at Biolog, Inc. (San Jose, CA) have developed a series of phenotype arrays that can be used to monitor and identify bacterial cell lines based upon their ability to grow and multiply in the presence of various nutrients, growth factors, inhibitors, hormones, drugs or other speciific compounds contained in the wells of the array. The amount of cell growth is monitored using a combination of tetrazolium dyes that are reduced by growing cells to form a purple formazan chromogenic signal. Cellular metabolism that is stimulated by the chemicals in the well generate reducing equivalents which are captured by the correlated instrumentation. Marker Gene has helped supply some of the reagents and detection systems for these array systems in the past. In addition, Biolog has recently developed similar phenotypic microarray systems for monitoring cell metabolism and growth of mammalian cells, that can be used in a high-throughput manner for drug development and cytotoxicity assays, monitoring energy metabolism pathways, comparing phenotypic changes caused by target gene inactivation, fingerprint analysis of cell types or other analyses. For more information about these techniques and methods, please see the references below or visit our website.

• Bochner BR, (2009) "Global Phenotypic Characterization of Bacteria." FEMS Microbiol. Rev. 33: 191-205.
• Y. Tohsato, H. Mori (2008) "Phenotype Profiling of Single Gene Deletion Mutants of E. coli Using Biolog Technology." Proceedings of the 19th International Conference on Genome Informatics (GIW 2008), Gold Coast, Australia, pp. 1-11.
• Johnson DA, Tetu SG, Phillippy K, Chen J, Ren Q, Paulsen IT, (2008) "High Throughput Phenotypic Characterization of Pseudomonas aeruginosa Membrane Transport Genes." PLoS Genet.4: 1-11.
• Burton, JD (2005) "The MTT assay to evaluate chemosensitivity." Methods in Molec. Med. 110: 69-78.

Firefly Luciferase Analysis in vivo.

Recently, the use of the luciferase marker gene in whole organisms has been developed, by combining luciferase transfection in specific tissues with systemic application of D-luciferin.  The ability to measure light emission from luciferase activity inside living tissues and in vivo by injecting live animals with D-luciferin (M0237) is aided by the use of cooled CCD or photon counting cameras that can easily analyze the low-light levels emitted in whole animal studies.  The potential of measuring tumor growth and tumor burden in vivo as well as the possibility of streamlining development of many types of therapies, including DNA-based gene therapies and gene vaccines using these new techniques is exciting. 

The recommended dosage for in vivo imaging with D-luciferin (M0237) is 150mg/kg via intraperitoneal (IP) administration.  This concentration works well across most species (mice, rat, rabbit, monkey, etc). In some cases, a lower dose may be sufficient, dependent upon experimental consistency.  In certain cases of limited sensitivity/low signal (e.g. poor transfection efficiency or expression levels) it can be advantageous to double the dose to 300mg/kg.  Doubling the dose, however, does not double the signal and even at 450mg/kg dosages it is difficult to reach saturation in vivo. But even at 450mg/kg, no toxicity has been observed. The effective D-luciferin concentration in cells and tissues becomes the limiting factor for signal intensity for in vivo settings, while in vitro, the cofactor ATP is the limiting reagent and must be added to in vitro assay systems. With intraperitoneal delivery, one creates a depot of substrate, which is slowly absorbed by the peritoneal vascularization and will freely diffuse across membranes into and out of all organs. D-Luciferin is not typically metabolized but excreted by the kidneys.

Intravenous (IV) injections in mice have been routinely performed in the tailvein or retroorbital plexus. Even though an IV injection is bolus, the same dose of 150mg/kg has been recommended. The maximal volume to inject IV is 10% of the blood volume. Since a 20g mouse is estimated to have 2ml blood, the maximal IV injection volume is about 200ul. Other methods of injection have included intranasal and subcutaneous methods.

Marker Gene has now developed a membrane permeable analog of D-luciferin, D-Luciferin, ethyl ester (M0906) to aide with these studies.  This analog of the primary substrate for the firefly luciferase light producing system has been found to produce up to 30% higher light intensity in vivo.  The esterification of the D-luciferin carboxyl group as an ethyl ester increases the uptake of the substrate by intact mammalian cells in live-cell luciferase assays.  After entering the cell, the ethyl ester is quickly removed by ubiquitous esterase activity inside living cells and tissues. For more information about these techniques, please see the references below or visit our website.

• Burgos JS, Rosol M, Moats RA, Khankaldyyan V, Kohn DB, Nelson MD, Laug WE. (2003) "Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice." Biotechniques 34(6):1184-8.
• Berger F, Paulmurugan R, Bhaumik S, Gambhir SS (2008) "Uptake kinetics and biodistribution of 14C-D-luciferin--a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging." Eur. J. Nucl. Med. Mol. Imaging. 35(12):2275-2278.
• Buckley SM, Howe SJ, Rahim AA, Buning H, McIntosh J, Wong SP, Baker AH, Nathwani A, Thrasher AJ, Coutelle C, McKay TR, Waddington SN (2008) " Luciferin detection after intranasal vector delivery is improved by intranasal rather than intraperitoneal luciferin administration." Hum. Gene Ther. 19(10): 1050-1056.
• Inoue Y, Kiryu S, Izawa K, Watanabe M, Tojo A, Ohtomo K (2009) "Comparison of subcutaneous and intraperitoneal injection of D-luciferin for in vivo bioluminescence imaging." Eur. J. Nucl. Med. Mol. Imaging 36(5): 771-779.

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