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Hypoxia in Tumor Tissues Measured Using Marker Genes
The absence of oxygen (hypoxia) is emerging as a potential new prognostic factor in the management of cancer. Tumors growing under hypoxic conditions have exhibited increased invasiveness, metastases, and resistance to therapy. Several genetic factors have been implicated in the tumors' ability to grow under the low oxygen conditions. Among the factors so far identified are hypoxia-inducible factor (HIF-1), nuclear factor kB and the hypoxia response element (HRE). In order to study the effect of these factors, gene constructs have been developed, linked to several reporter genes, to monitor their expression levels both in vivo and in vitro. But new information from the laboratory of Dr. Edward E. Graves and co-workers at the Department of Radiation Oncology and Molecular Imaging, Stanford University School of Medicine have indicated that the marker genes themselves may be sensitive to low oxygen environments. They investigated the oxygen sensitivity of four commonly used marker genes: Renilla luciferase (luc), red fluorescent protein (RFP), thymidine kinase (TK), and lacZ beta-galactosidase. The relationship between the actual level of reporter protein produced to the reporter signal generated, as a function of oxygen levels was assessed for each marker gene both in cell culture and live mouse assay conditons. Interestingly, the fluorescence or luminescence signal for the luciferase, RFP and TK genes were significantly reduced under low oxygen conditions compared to the actual protein levels expressed. But the fluorescence levels for lacZ beta-galactosidase using the galactosidase substrate DDAOG, a resorufin beta-D-galactoside derivative, were unaffected by the hypoxic conditions which occur both in tumors and also in stroke and myocardial infarction conditions. For more information about these assays and substrates, please see the references below or visit our website.

• Cecic I, Chan DA, Sutphin PD, Ray P, Gambhir SS, Giaccia AJ, Graves EE (2007) "Oxygen Sensitivity of Reporter Genes: Implications for Preclinical Imaging of Tumor Hypoxia." Molecular Imaging 6(4): 219-225.
• Bhaumik S, Lewis XZ, Gamhhir SS (2004) "Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice. J. Biomed Opt. 9:578-586.
• Corelli C, Cemazar M, Kanthou C, Tozer GM, Dachs GU, (2001) "Limitations of the reporter green fluorescent protein under simulated tumor conditions." Cancer Res 61:4784-90.

Transgenic Feline Model with Ubiquitous Red Fluorescent Protein Expression.

Methods for homologous gene transfer into mammalian species are of interest for use in gene therapy and development of disease models for a variety of disease states. Recently, researchers from the laboratory of Dr. Il Keun Kong and co-workers at Gyeongsang National University, Jinju, South Korea have successfully cloned the Red Fluorescent Protein (RFP) into Turkish Angora cats at the embryo stage providing ubiquitous marker gene expression in all tissues. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses 60 days after birth. Whole body red fluorescence was detected in the liveborn transgenic cats, but not in the surrogate mother. Red fluorescence was detected in tissue samples including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, and tongue of these transgenic cats. These results suggest that the nuclear transfer procedure using genetically modified somatic cells could be useful for more efficient production of other transgenic species. The marker gene was used as a method of improving the efficiency of transgenic animal production by screening for genetically modified embryos prior to transfer into surrogate mothers.

The group utilized a technique call somatic cell nuclear transfer (SCNT) to produce the cloned cats and transfer the RFP gene into the cat embyros. This SCNT technique has already been used to produce transgenic cattle, sheep, goats, mice and pigs. But the success rate for growth to adult animals has thrusfar been very low. The technique involves removing the nucleus of an unfertilized egg cell and replacing it with the nucleus of a "somatic cell" (in this case, a skin cell), and then stimulating this cell to begin dividing and growing. Donor fibroblasts were obtained from an ear-skin biopsy of a white male Turkish Angora cat. After culturing for one to two passages, they were then subjected to transduction with a retroviral vector containing the red fluorescent protein (RFP) gene. For more information about these new labeling techniques, please see the references below, or contact our technical assistance department at techservice@markergene.com.

• Yin XJ, Lee HS, Yu XF, Choi E, Koo BC, Kwon MS, Lee YS, Su Jin Cho SJ, Jin GZ, Kim LH, Shin HD, Kim T, Kim NH, Il Keun Kong IK (2007) "Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein."
• Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998) " Cloned transgenic calves produced from nonquiescent fetal fibroblasts." Science 280: 1256-1258.
• Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King T, Ritchie M, Ritchie WA, Rollo M, de Sousa P, Travers A, Wilmut I, Clark AJ. (2001) "Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep." Nature Biotechnology 19: 559-562.
• McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE, Kind AJ. (2000) "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405: 1066-1069.
• Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris A, Karatzas CN. (2001) "Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes." Biology of Reproduction 64: 849-856.
• Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL. (2002) "Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs." Nature Biotechnology 20: 251-255.
• Lai L, Prather RS. (2002) "Progress in producing knockout models for xenotransplantation by nuclear transfer." Annals of Medicine 34: 501-506.
• Sato K, Hosaka K, Ohkawa M, Tokieda Y, Ishiwata I. (2001) "Cloned transgenic mouse fetuses from embryonic stem cells." Human Cell 14: 301-304.

Carbohydrate Analysis by Fluorescence Derivatization.

• Jackson, P., (1996) “The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis.” Mol Biotechnol. 5(2): 101-23.
• Morimoto K., Maeda N., Abdel-Alim A.A., Toyoshima S., Hayakawa T., (1999) “Structural characterization of recombinant human erythropoietins by fluorophore-assisted carbohydrate electrophoresis.” Biol. Pharm. Bull. 22(1): 5-10.
• Cottaz S., Brasme B., Driguez H., (2000) “A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.” Eur. J. Biochem. 267(17): 5593-5600.
• Rago, RP, Ramirez-Soto, D, Poretz, RD, (1992) "Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans. Carbo. Res. 236: 1–8.
• Harvey DJ, Wing DR, Kuster B, Wilson IBH, (2000) "Composition of N-Linked Carbohydrates from Ovalbumin and Co-purified Glycoproteins." J. Amer. Soc. Mass Spectrom. 11: 564–571.
• Wu W, Hamase K, Kiguchi M, Yamamoto K, Zautsu K (2000) "Reversed Phase HPLC of Monosaccharides in Glycoproteins Derivatized with Aminopyrazine with Fluorescence Detection." Anal. Sciences 16: 919-922.

Mitochondrial and Cellular Membrane Potential Measurements Using DiS-C3(3).
The mitochondria are the powerhouses of the cell and their respiratory chain plays a crucial role in cellular physiology and health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis, the generation of reactive oxygen species and cellular potential. Monitoring changes in the cellular and mitochondrial membrane potential (ΔΨm) in vivo has been accomplished in several species by staining with the membrane potential sensitive dye DiS-C3(3), 3,3'-Dipropylthiacarbocyanine iodide (M1331). This dye can exhibit fluorescence changes of up to 80% upon membrane hyperpolarization (where the inside of the cell becomes more negative). Its fluorescence emission and excitation (λex = 556 nm; λem =  575 nm in MeOH; λex = 520, λem = 595nm in water) are in the red region of the visible spectrum, allowing co-staining with other viability stains. Cell hyperpolarization results in an uptake of the dye molecules by the cells, while depolarization results in release of the dye. The emission from cell-associated dye loading becomes significantly quenched as the amount of intracellular dye increases. The quenching of dye is most likely due to the formation of dye aggregates. The intensity of fluorescence of stained cells increases proportionally to probe concentration over the range of about 60-3000 nanomolar. An optimum staining period is about 15-20 min has been found for labeling with diS-C3(3). Depolarization of cells by increasing extracellular potassium levels or by addition of valinomycin can be used to elicit a drop in fluorescence intensity.

In yeast, the redistribution of diS-C3(3) responds to yeast plasma membrane depolarisation or hyperpolarisation (outflow or uptake into the cells). These are also reflected in changes in the fluorescence λmax wavelength as well as fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to Δψ-independent fluorescence responses. They may also exhibit Δψ-dependent blue or red shifts in fluorescence emission wavelengths (λmax). These λmax shifts after cell permeabilisation are also dependent on probe and ion concentrations inside and outside of the cells. For more information on these mitochondrial staining methods, please see the references below, or visit our website.

• Gaskova D, DeCorby A, Lemire BD, (2007) "DiS-C3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans." Biochemical and Biophysical Research Communications 354(3): 814-819.
• Sims PJ, Waggoner AS, Wang CH, Hoffman JF, (1974) "Mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles." Biochemistry13(16): 3315-30.
• Okimasu E, Kobayashi S, Aono K, Tomoda T, Utsumi K, (1981) "Tests of fluorescence changes in tumor cells due to changes in mitochondria energy metabolism with low-toxicity NK 1507." Kanko Shikiso 88: 21-30.
• Denksteinova B, Sigler K, Plasek, J. 1996) "Three fluorescent probes for the flow-cytometric assessment of membrane potential in Saccharomyces cerevisiae." Folia Microbiologica (Prague) 41(3): 237-242.
• Vecer J, Herman P, Holoubek A, (1997) "Diffusion membrane potential in liposomes: setting by ion gradients, absolute calibration and monitoring of fast charges by spectral shifts of diS-C3(3) fluorescence maximum." Biochimica et Biophysica Acta, Biomembranes 1325(2): 155-164.
• Herman P, Vecer J, Gaskova D, Denksteinova B, Holoubek A, Plasek J, Sigler K, (1998) "Assessment of mitochondrial potential in yeasts by the fluorescent dye diS-C3(3): monitoring and modeling of membrane potential transients. Fluorescence Microscopy and Fluorescent Probes" [Based on the Proceedings of the Conference on Fluorescence Microscopy and Fluorescent Probes], 2nd, Prague, Apr. 9-12, 1997 (1998), Meeting Date 1997, 141-145.
• Gaskova D, Cadek R, Chaloupka R, Plasek J, Sigler K, (2001) "Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast." Biochimica et Biophysica Acta, Biomembranes 1511(1): 74-79.

Visit us at the Experimental Biology and Cell Biology Meetings.
Marker Gene will be exhibiting and presenting several posters on our latest research at the following meetings in 2008.

April 5-9: Experimental Biology, San Diego, CA (www.faseb.org/meetings)

December 13-17: American Society for Cell Biology, San Francisco, CA (www.ascb.org)

Please stop by to discuss your research and development efforts. For updates, please visit our website at http://www.markergene.com/. We appreciate your input for new products and methods that are of interest to the worldwide scientific community. We also develop products for specific applications and for exclusive uses.

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 details about our products and their specifications.

• 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.

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