Marker Gene Monthly Newsletter   

September, 2006

Volume 6 , Number 9

© 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.

hGH Reporter Gene Assays.

The human Growth Hormone (hGH) marker gene system is an older marker gene method that utilizes expression of the hGH protein (21.5kD) that is secreted into the culture medium by the transfected cells. Since hGH is a secreted protein, it can be easily measured using aliquots of culture-medium supernatants thus avoiding the necessity to lyse cells. This also allows for continuous monitoring of expression kinetics, and the cells in culture can be maintained growing for other purposes. It has also been found that since hGH is a mammalian gene, it has a higher stability in most mammalian cell lines and there is often a substantially greater accumulation of hGH mRNA intracellularly than with other reporter genes . Q uantifying the reporter protein, however, is somewhat laborious and is usually carried out using 125 I-labeled antibodies against the growth hormone. There are also several non-radioactive alternative ELISA assays. In these systems, anti-hGH antibodies are bound to the surface of a microtiter plate. The hGH from the supernatant of the culture medium binds to the antibody on the plate. After washing, the bound hGH is detected in two steps via a digoxigenin- coupled anti-hGH antibody (anti-hGH-DIG) and a peroxidase-coupled anti-digoxigenin antibody (anti-DIG-POD) . Bound peroxidase is quantified by incubation with a chromogenic substrate like TMB (3,3',5,5'-tetramethylbenzidine) or ABTS ( 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) , yielding a blue or blue-green colored product, respectively. Based on ease of detecting expression from various comparable plasmids, the hGH assay seems to be a slightly more sensitive than the CAT assay. This is at least partly a consequence of the substantially greater accumulation of hGH mRNA.

Quantitation of hGH secretion is typically performed by radioimmunoassay with a Nichols Diagnostic kit. However, these kits have recently become unavailable commercially from Nichols, but other companies have similar assay systems. All hGH measurements are typically normalized to total cell protein determined by Bradford protein assay. This assay is often used in co-transfection experiments to normalize for transfection efficiency, and for routine analysis of gene expression levels based upon promoter activation. For example, the hGH reporter gene system has been used in a high-throughput method to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction by monitoring increases in hGH secretion following stimulation by phorbol esters, mitogens or other pharmacological agents. Recently a monoclonal antibody specific for hGH has been described which can also be used for immunohistochemical analysis of hGH levels in cells and tissues. For more information about this reporter gene system, please visit our website , or see the references below:

• Sacco * , M.G., Zecca, L., Bagnasco L., Chiesa G., Parolini, C., Bromley, P., Catò, E.M., Roncucci, R., Clerici L.A., Vezzoni P., (1997) “ A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds.” Nature Biotechnology   15: 1392 - 1397
• Selden, R. F., Howie, B., Rowe, E., Goodman H.M., Moore D.D., ( 1986) “Human growth hormone as a reporter gene in regulation studies employing transient gene expression.” Mol Cell. Biol. 6(9): 3173-3179.
• Bennani-Baiti, I. M. , Asa, S. L. , Song, D. , Iratni, R. , Liebhaber, S. A., Cooke, N. E. (1998) “DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression” Proc. Natl. Acad. Sci. USA 95: 10655-10660.
• Sista P., Edmiston S., Darges J.W., Robinson S., Burns D.J.,  (1994) “A cell-based reporter assay for the identification of protein kinase C activators and inhibitors.” Mol. Cell Biochem. 41(2): 129-34
• Khorshed S. M. Alam, Takahiko Fujikawa, Hideo Yoshizato, Minoru Tanaka and Kunio Nakashima (2000) “ Synthesis and purification of a deleted human growth hormone, hGH ? 135–146 : sensitivity to plasmin cleavage and in vitro and in vivo bioactivities” Journal of Biotechnology 78(1): 49-59.
• Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl (1992) In: Current protocols in molecular biology; publ. John Wiley & Sons, Vol. 1, suppl. 14, 9.7.
  

Temperature Dependence of Fluorescence.

The fluorescence of common dyes including fluorescein, rhodamine and eosin can be affected by changes in temperature, regardless of other biological characteristics involved in their applications. In general, quantum yield, and thus fluorescence intensity both increase as temperature decreases, and, in polar solvents, the effect is even more pronounced. In addition, excited-state lifetimes of fluorescent molecules are also affected by temperature, showing, in most cases, longer lifetimes at lower temperatures. This effect of increasing fluorescence lifetimes seems to be largely due to a decrease in the rates of nonradiative decay at lower temperatures.

There are also slight shifts in the wavelengths of emission and excitation maxima that occur with temperature changes. When observed, the temperature shift of the peak of the fluorescence bands is proportional to the change in temperature. The observed changes are in the same direction as those observed for absorption. But the shift is not in the same direction for all substances. For Rhodamine B, for example, decreases in temperature or increases in concentration cause the peaks of the bands to shift to longer wavelengths. Decreases in concentration cause a widening of the fluorescence band, and a narrowing of the absorption band. For Eosin a decrease in temperature causes a shift to shorter wavelengths for fluorescence as well as for absorption. Concentration shifts are typically minimal. Eosin fluorescence is at a maximum at or near room temperature. Fluorescein exhibits a shift toward shorter wavelengths as temperature decreases. It's fluorescence also is at a maximum at or near room temperature. Further increases in temperature cause a widening of both the fluorescence and the absorption bands, and this widening is greater on the red side of band. There are also wavelength shifts that occur due to changes in concentration, which are proportional to the logarithm of the concentration. Again the observed shift will be in the same direction as it is for absorption. But changes in the width of the fluorescence band do not follow the same law as for absorption. In general, the resulting wavelength shifts caused by changes in temperature or changes in concentration seem to be the same for absorption as for fluorescence. For more information about these physical properties of fluorophores, please visit our website or see the references below:

• Larry D. Faller, Ruben A. Diaz, Georgios Scheiner-Bobis, and Robert A. Farley (1991) “ Temperature Dependence of the Rates of Conformational Changes Reported by Fluorescein 5'-Isothiocyanate Modification of H+,K+- and Na+,K+-ATPases” Biochemistry 1991, 30: 3503-3510.
• Giri R. (1992) “Temperature effect study upon the fluorescence emission of substituted coumarins” Spectrochim. acta, Part A : Mol. spectrosc. 48(6):  843-848.
• Ann E. Oliver, Gary A. Baker, Robert D. Fugate, Fern Tablin, and John H. Crowe (2000) “Effects of Temperature on Calcium-Sensitive Fluorescent Probes” Biophysical Journal 78: 2116–2126.
• Jay R. Unruh, Giridharan Gokulrangan, George S. Wilson, Carey K. Johnson (2005) “Fluorescence Properties of Fluorescein, Tetramethylrhodamine and Texas Red Linked to a DNA Aptamer” Photochemistry and Photobiology 81(3): 682–690.
• J. R. Jenness (1929) “ Effect of Temperature Upon the Fluorescence of some Organic Solutions” Phys. Rev. 34(9): 1275–1285.
• Eastman, J. W., and E. J. Rosa. (1968) “The fluorescence of adenine. The effects of solvent and temperature on the quantum yield.” Photochem. Photobiol. 7: 189 –203.
• Song, P.-S., Q. Chae, and W. R. Briggs. (1975) “Temperature dependence of the fluorescence quantum yield of phytochrome.” Photochem. Photobiol. 22: 75–76.
• Cornelissen-Gude, C., and W. Rettig. (1998) “Temperature dependence of the multiple fluorescence of 9,9-dianthrylmethanol.” Chemical Physics. 229: 325–334.
• Haynes, D. R., A. Tokmakoff, and S. M. George. (1993) “Temperature-dependent absolute fluorescence quantum yield of C60 multilayers.” Chem. Phys. Lett. 214: 50 –56.
• Connors, R., V. Chynwat, C. H. Clifton, and T. L. Coffin. (1998) “Temperature dependence of aryl butatriene fluorescence: barrier to twisting on S1 for 1,1,4,4-tetrapeynylbutatriene.” J. Molec. Struct. 443: 107–113.
• Park, T.-R. (1996) “The temperature dependence of the fluorescence inten-sity and a nonradiative de-excitation process in sodium cryptand sodide.” J. Phys.: Condens. Matter. 8: 405– 418.
• Law, K.-Y. (1994) “Squaraine chemistry: effects of solvent and temperature on the fluorescence emission of squaraines. J. Photochem. Photobiol. A: Chemistry.” 84: 123–132.
• Waris, R., W. E. Acree, and K. W. Street . (1988) “Py and BPe solvent polarity scales: effect of temperature on pyrene and benzo[ghi]perylene fluorescence spectra.” Analyst. 113: 1465–1467.

Generic Expression Vectors

Marker Gene now provides several generic expression vectors that can be used to express common reporter genes in your cell lines. These eukaryotic expression vectors are very useful for transfection of mammalian cells in culture, or for use in other species. Several vectors expressing the full-length b -galactosidase gene are available, with b -Gal enzyme expression enhanced by elements including: SD/SA-RNA splice donor and acceptor sequence, and SV40 late polyadenylylation signal. Expression in our pSV40 b Mammalian lacZ vector (Product M0952 ) is under the control of simian virus 40 (SV40) early promoter. Expression in our pCMV b Mammalian lacZ vector (Product M0951 ) is under the control of the cytomegalovirus immediate early gene (CMV IE) promoter. pCMV b is a high copy number vector that has been tested to generate up to 2530 u/mg cell extract. These vectors also contain several convenient restriction sites that allow the b -galactosidase gene to be easily excised or to allow insertion of other genes to be expressed under the same regulatory elements in mammalian cells. In addition, both vectors encode b -lactamase, which acts as a selection marker (100 m g/mL ampicillin resistance) in an E. coli host, for vector amplification protocols. For more information on these expression vectors, please visit our website (at http://www.markergene.com ) or see the references below.

• Hall, C.V. Jacob P.E., Ringold G.M., Lee F.,  ( 1983) “Expression and Regulation of Escherichia coli lacZ Gene Fusions in Mammalian Cells.” J. Mol. Appl. Gen. 2 :101.
• Herbomel, P. Bourachot B., Yaniv M., ( 1984) “ Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. ” Cell 39 :653.
• Nikcevic, G. Kovacevic-Grujicic N., Stevanovic M.,  (2003) “ Improved transfection efficiency of cultured human cells. ” Cell Biol. Int. 27 :735.
• MacGregor, G.R., Caskey, C.T., (1989) “Construction of Plasmids that Express E. Coli Beta-galactosidase in Mammalian Cells.” Nucleic Acids Res. 17 : 2365.
• Norton, P.A., Coffin, J.M., (1984) “Bacterial b -galactosidase as a Marker of Rous Sarcoma Virus Gene Expression and Replication.” Mol. Cell Biol. 5 : 281.
• Nolan, G.P., Fiering S., Nicolas J.F., Herzenberg L.A., (1988) “Fluorescence-activated Cell Analysis and Sorting of Viable Mammalian Cells Based on b -D-galactosidase Activity After Transduction of Escherichia coli lacZ.” Proc. Natl. Acad. Sci ( USA ). 85 : 2603.

Efficient lacZ FACS Analysis in Bacteria.

One of the most common reporter genes used in molecular biology applications is the E. coli lacZ gene, which codes for an active subunit of b -galactosidase in vivo . Expression of lacZ can be quantitated by measuring the resulting intracellular b -galactosidase activity. b -galactosidase activity assays can be performed in vivo without cell ablation, using the non-toxic fluorogenic substrate fluorescein di- b -D-galactopyranoside ( FDG , M0250 ). FDG is an extremely sensitive indicator of b -galactosidase activity, several magnitudes more sensitive than chromogenic substrates such as X-Gal and ONPG, and allows expression as low as five copies per cell to be quantitated using fluorescence activated cell sorting (FACS) systems. We have now adapted our popular MarkerGene TM in vivo lacZ b -galactosidase Detection Kit ( Product M0259 ) for measurement of b -galactosidase activity using FACS in bacteria and yeast cells, in addition to mammalian cells. This FACS analysis of lacZ expression in bacterial cells can be performed using an quick and simple procedure (see below) that can be performed in less than one hour. The kit contains enough reagents to run multiple assays, as well as a detailed protocol and additional reagents for quantitation of the results.

FACS analysis of Bacterial lacZ expression (suggested protocol, see http://www.markergene.com/product_sheets/pis0259a.pdf for more information)

• Pellet bacterial cells from liquid culture at 10,000 x g.
• Resuspend cells in distilled water (1mL).
• Add FDG substrate reagent (product M0259-001 ) to cell suspension so that final FDG concentration is 1mM.
• Incubate cells at 37°C for 30 minutes.
• Perform FACS analysis as per manufacturer's instructions.
  For FACS analysis of lacZ expression in yeast, the cells must first be permeabilized. Please see the MarkerGene TM In vivo lacZ b -galactosidase Detection Kit product page ( http://markergene.com/product_sheets/0259.htm ) and the following references for more information regarding applications of the kit for analysis of bacterial and yeast cell lacZ expression.
• Plovins A., Alvarez A., Ibanez M., Molina M., Nombela C. (1994) “Use of Fluorescein-Di-ß-D-Galactopyranoside (FDG) and C12-FDG as Substrates for ß-Galactosidase Detection by Flow Cytometry in Animal, Bacterial, and Yeast Cells.” Appl Environ Microbiol. 60(12): 4638-4641.
• Fierer J., Eckmann L., Fang F., Pfeifer C., Finlay B.B., Guiney D. (1993) “Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells.” Infection and Immunity 61(12): 5231-6
• Roederer M., Fiering S., Herzenberg L.A. (1991) "FACS-Gal: flow cytometric analysis and sorting of cells expressing reporter gene constructs." Methods: Comp. Meth. Enzymol. 2 : 248.
  
  

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

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