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Marker Gene Monthly Newsletter

November, 2005

Volume 5, Number 11

© 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.

Circadian Rhythms Monitored Using an mPer2 Luciferase Construct.

Circadian Rhythms (often termed the biological clock) control a variety of biological functions in organisms by genetic control of gene expression and physiology. Recent work from the laboratory of David K. Welsh and Steven Kay at the Department of Cell Biology, Scripps Research Institute, have been able to monitor the circadian rhythms in individual SCN mammalian cells using a mPer2 promoter linked to the firefly luciferase gene, with addition of the substrate D-Luciferin (M0237). This assay monitored chemiluminescent light emission as a function of time. Using a “knock-in” mutant approach, homologous recombination was used to insert into the circadian clock gene mPer2, a luciferase gene under control of the mPer2 promoter. The circadian phenotype of the new “knock-in” cells was normal, without detectable effect on the tertiary structure of the mPer2 protein. As expression of the mPer2 protein increased (on a 24-hour cycle) so did the output of light. A landmark paper was also recently published by Yamaguchi, et al. using a similar mPer1-luciferase construct for single-cell bioluminescence imaging. These assays have revealed new features of the cellular organization of the central mammalian circadian clock. Because the light intensity for these assays are so low, the cell images required 30 minute exposures using a high-sensitivity CCD camera to monitor the changes.

The SCN cells come from the suprachiasmatic nucleus, a tiny cluster of neurons located in the hypothalamus that is thought to be the “clock pacemaker” of the brain. SCN cells in culture have been found to maintain their circadian rhythms. The same clock genes have been found to be expressed rhythmically in fibroblasts and other cell types, but it is not known if this rhythm is driven by an internal clock or merely synchronized externally. Fibroblasts in culture maintain circadian rhythms that persist for about a day, but then begin to decrease in amplitude. For more information about luciferase assays and circadian rhythm analyses, please see the references below, or visit our website.

Welsh D..K., Imaizumi T., Kay S.A., (2005) “Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and Mammalian cells.” Methods Enzymol 393: 269-88.

Welsh D.K., Yoo S.H., Liu A.C., Takahashi J.S., Kay S.A., (2004) “Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.” Curr Biol 14(24): 2289-95.

·Welsh D.K., Kay S.A., (2005) “Bioluminescence imaging in living organisms.” Curr. Opin. Biotechnol. 16(1): 73-8.

Welsh D.K., Logothetis DE., Meister M., Reppert S.M., (1995) “Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms.” Neuron 14(4): 697-706.

Yamaguchi S., Isejima H., Matsuo T., Okura R., Yagita K., Kobayashi M., Okamura H., (2003) “Synchronization of cellular clocks in the suprachiasmatic nucleus.” Science, 302:1408-1412.

Viability Assays for Eukaryotic Cells.

Cell viability assays are commonly used to assess the effects of secondary reagent application or the physiological state of eukaryotic cells. These assays include those based on the generation of the colorimetric end product, formazan. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is based on the ability of metabolically active cells to cleave the tetrazolium rings of MTT, forming dark blue formazan crystals within the cell. The cells are then lysed and the formazan crystals are solubilized with detergent. The resulting color can be quantified using a standard spectrophotometer, reading absorbance at 570 nm. The number of live cells is directly proportional to the intensity of the dye. XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) is an alternative tetrazolium salt. Unlike MTT, the XTT formazan dye is water soluble and does not require a solubilization step. Absorbance is measured at 490 nm. Fluorimetric assays based on compounds such as calcein AM and carboxyfluorescein diacetate (CFDA) (M0011) can also be used to determine cell viability. These molecules passively diffuse into cells, but only live cells with functional esterase activity will hydrolyze the molecules, producing the fluorescent dyes calcein and carboxyfluorescein. Both dyes will stain the cytoplasm of the cells. Calcein has peak absorbance and emission wavelengths at 494 nm and 517 nm, respectively. Carboxyfluorescein has peaks at 475 nm and 517 nm. Marker Gene also sells a convenient Live:Dead Assay Kit (M0795) that contains both carboxyfluorescein for labeling live cells and propidium iodide, which labels dead cells red. For more information about these assays and techniques, please visit our website or see the references below:

T. Mosmann (1983) “Rapid colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays” J. Immunol. Meth. 65(1-2): 55-63.

D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofiziger, M.J. Currens, D. Seniff, M.R. Boyd (1988) “Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using human and other Cell Lines” Cancer Res. 48: 4827-4833.

N.R. Washburn, C.G. Simon Jr, A. Tona, H.M. Elgendy, A. Karim, E.J. Amis (2002)“Co-extrusion of Biocompatible Polymers for Scaffolds with Co-continuous Morphology” J. Biomed. Mater Res. 60: 20-29.

C.R. Parish (1999) “Lymphocyte Migration and Proliferation Studies” Immunol. Cell Biol. 77:499-508.

Lipase Activity Measurement in Live Cells.

Triacylglycerides are metabolized inside living cells by lipases [EC 3.1.1.3]. In normal serum the concentration of lipase is low. In acute pancreatitis and in pancreatic carcinoma a rise in serum (pancreatic) lipase activity occurs, with a mean increase being about 50 times that of normal values. A rise in the serum lipase (hepatic lipase) content is also found in acute and chronic renal diseases. Sensitive measurement of lipase activity in live cells can be accomplished using the fluorescent substrate 1,2-Dioleoyl-3-(pyren-1-yl)decanoyl-rac Glycerol (M0258). Upon enzymatic cleavage, the fluorescent fatty acid, pyrenedecanoic acid (M0274) is released, which accumulates in cellular membranes. The substrate displays eximer formation, and upon enzymatic activity, there is a fluorescence shift to shorter wavelengths. This can be distinguished from that of the substrate, because of the reduction of the eximer form inside the membrane (EM: 470nm @ EX: 390nm). This assay is quantitative and can be used with mammalian or bacterial cell lines. It can also be used in a purified enzyme format with the addition of lipid vesicles or added membrane components. This assay has also found use in measurement of endothelial lipases (EL) and lipoprotein lipase (LPL). For more information about this assay see the references below: NOTE: Marker Gene also sells a convenient Fluorescent Lipase Assay Kit (M0612) with all of the reagents and a detailed protocol for measuring lipase activity in living cells.

Dousset, N., Negre, A., Salvayre, R., Rogalle, P., Dang, Q.Q., Douste-Blazy, L. (1988). “Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase.” Lipids 23: 605-608.

Main, L.A., Okumura-Noji, K., Ohnishi, T., Yokoyama, S., (1998) “Cholesteryl ester transfer protein reaction between plasma er R., (2000) “Methods for lipase detection and assay: a critical review” Eur. J. Lipid Sci. Technol.2000: 133–153.

lipoproteins.” J. Biochem. (Tokyo) 124: 237-243.

Negre-Salvayre, A., Abouakil, N., Lombardo, D., Salvayre, R., (1990) "Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase." Lipids 25(8): 428-434.

Beisson F., Tiss, A., Rivière C., Verg

Stock up and Save!
Marker Gene will introduce a price increase of 5% on most products and kits from our catalog, starting on January 1^st, 2006. Orders placed and received before January 1^st will still be credited for our 2005 prices. So place your orders now, online, through one of our distribution partners, by FAX (1-541-342-1960) or by telephone (toll-free) at 1-888-218-4062, and save. For more information on the new 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.

New lacZco Vector Systems.

Marker Gene offers a variety of new vectors with improved expression profiles for the lacZ (b-galactosidase) gene (M1015, pCMVblacZco; M1016, pSV40blacZco, M1017 pCMVblac Znls12co and M1018 pSV40blac Znls12co). These vectors contain a coding sequence for the E. coli lacZ gene that has been codon-optimised when expressed in mammalian cells. This results in the expression of b-galactosidase at levels up to 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of b-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. The nls12 sequence variant also targets the protein for nuclear localization by the addition of a twelve amino acid sequence, ProLysLysLysArgLysValGluAspProLysAsp (from the SV40 T antigen nuclear localization signal) after the methionine initiation residue.

Marker Gene is currently working with Dr. Donald Anson at the Department of Chemical Pathology, Women's and Children's Hospital in North Adelaide, Australia to also provide newly developed lacZ expression lentiviral vectors with high expression efficiency in mammalian cells. When used in such a lentiviral construct, the codon-optimised gene gives an approximately five-fold increase in apparent titre, as determined by X-Gal staining, in comparison to an analogous construct containing the native E. coli gene. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised b-galactosidase gene has a number of advantages over the native E. coli gene sequence. For more information on these new vector systems, see the references below or visit our website. Look for these new vectors in kits from Marker Gene soon!

Anson D.S., Limberis M., (2004) “An improved beta-galactosidase reporter gene.” J Biotechnol 108(1): 17-30.

http://biology.kenyon.edu/courses/biol09/betagal/FRAMES/

MacGregor, G. R. & Caskey, C. T. (1989) “Construction of plasmids that express E. coli beta-galactosidase in mammalian cells.” Nucleic Acids Res. 17:2365.

Okayama, H. & Berg, P. (1983) “A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.” Mol. Cell Biol. 3:280-289.

MacGregor, G. R.,et al. (1987) “Histochemical staining of clonal mammalian cell lines expressing E. coli beta-galactosidase indicates heterogeneous expression of the bacterial gene.” Somat. Cell Mol. Genet. 13:253-265.

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.


	If you are having trouble viewing this email, click on this link. To order any of our products, please go to our ORDER FORM or visit one of our distributors:
	Marker Gene Monthly Newsletter November, 2005 Volume 5, Number 11 © 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.
	Circadian Rhythms Monitored Using an mPer2 Luciferase Construct. Circadian Rhythms (often termed the biological clock) control a variety of biological functions in organisms by genetic control of gene expression and physiology. Recent work from the laboratory of David K. Welsh and Steven Kay at the Department of Cell Biology, Scripps Research Institute, have been able to monitor the circadian rhythms in individual SCN mammalian cells using a mPer2 promoter linked to the firefly luciferase gene, with addition of the substrate D-Luciferin (M0237). This assay monitored chemiluminescent light emission as a function of time. Using a “knock-in” mutant approach, homologous recombination was used to insert into the circadian clock gene mPer2, a luciferase gene under control of the mPer2 promoter. The circadian phenotype of the new “knock-in” cells was normal, without detectable effect on the tertiary structure of the mPer2 protein. As expression of the mPer2 protein increased (on a 24-hour cycle) so did the output of light. A landmark paper was also recently published by Yamaguchi, et al. using a similar mPer1-luciferase construct for single-cell bioluminescence imaging. These assays have revealed new features of the cellular organization of the central mammalian circadian clock. Because the light intensity for these assays are so low, the cell images required 30 minute exposures using a high-sensitivity CCD camera to monitor the changes. The SCN cells come from the suprachiasmatic nucleus, a tiny cluster of neurons located in the hypothalamus that is thought to be the “clock pacemaker” of the brain. SCN cells in culture have been found to maintain their circadian rhythms. The same clock genes have been found to be expressed rhythmically in fibroblasts and other cell types, but it is not known if this rhythm is driven by an internal clock or merely synchronized externally. Fibroblasts in culture maintain circadian rhythms that persist for about a day, but then begin to decrease in amplitude. For more information about luciferase assays and circadian rhythm analyses, please see the references below, or visit our website. Welsh D..K., Imaizumi T., Kay S.A., (2005) “Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and Mammalian cells.” Methods Enzymol 393: 269-88. Welsh D.K., Yoo S.H., Liu A.C., Takahashi J.S., Kay S.A., (2004) “Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.” Curr Biol 14(24): 2289-95. ·Welsh D.K., Kay S.A., (2005) “Bioluminescence imaging in living organisms.” Curr. Opin. Biotechnol. 16(1): 73-8. Welsh D.K., Logothetis DE., Meister M., Reppert S.M., (1995) “Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms.” Neuron 14(4): 697-706. Yamaguchi S., Isejima H., Matsuo T., Okura R., Yagita K., Kobayashi M., Okamura H., (2003) “Synchronization of cellular clocks in the suprachiasmatic nucleus.” Science, 302:1408-1412.
	Viability Assays for Eukaryotic Cells. Cell viability assays are commonly used to assess the effects of secondary reagent application or the physiological state of eukaryotic cells. These assays include those based on the generation of the colorimetric end product, formazan. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is based on the ability of metabolically active cells to cleave the tetrazolium rings of MTT, forming dark blue formazan crystals within the cell. The cells are then lysed and the formazan crystals are solubilized with detergent. The resulting color can be quantified using a standard spectrophotometer, reading absorbance at 570 nm. The number of live cells is directly proportional to the intensity of the dye. XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) is an alternative tetrazolium salt. Unlike MTT, the XTT formazan dye is water soluble and does not require a solubilization step. Absorbance is measured at 490 nm. Fluorimetric assays based on compounds such as calcein AM and carboxyfluorescein diacetate (CFDA) (M0011) can also be used to determine cell viability. These molecules passively diffuse into cells, but only live cells with functional esterase activity will hydrolyze the molecules, producing the fluorescent dyes calcein and carboxyfluorescein. Both dyes will stain the cytoplasm of the cells. Calcein has peak absorbance and emission wavelengths at 494 nm and 517 nm, respectively. Carboxyfluorescein has peaks at 475 nm and 517 nm. Marker Gene also sells a convenient Live:Dead Assay Kit (M0795) that contains both carboxyfluorescein for labeling live cells and propidium iodide, which labels dead cells red. For more information about these assays and techniques, please visit our website or see the references below: T. Mosmann (1983) “Rapid colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays” J. Immunol. Meth. 65(1-2): 55-63. D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofiziger, M.J. Currens, D. Seniff, M.R. Boyd (1988) “Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using human and other Cell Lines” Cancer Res. 48: 4827-4833. N.R. Washburn, C.G. Simon Jr, A. Tona, H.M. Elgendy, A. Karim, E.J. Amis (2002)“Co-extrusion of Biocompatible Polymers for Scaffolds with Co-continuous Morphology” J. Biomed. Mater Res. 60: 20-29. C.R. Parish (1999) “Lymphocyte Migration and Proliferation Studies” Immunol. Cell Biol. 77:499-508.
	Lipase Activity Measurement in Live Cells. Triacylglycerides are metabolized inside living cells by lipases [EC 3.1.1.3]. In normal serum the concentration of lipase is low. In acute pancreatitis and in pancreatic carcinoma a rise in serum (pancreatic) lipase activity occurs, with a mean increase being about 50 times that of normal values. A rise in the serum lipase (hepatic lipase) content is also found in acute and chronic renal diseases. Sensitive measurement of lipase activity in live cells can be accomplished using the fluorescent substrate 1,2-Dioleoyl-3-(pyren-1-yl)decanoyl-rac Glycerol (M0258). Upon enzymatic cleavage, the fluorescent fatty acid, pyrenedecanoic acid (M0274) is released, which accumulates in cellular membranes. The substrate displays eximer formation, and upon enzymatic activity, there is a fluorescence shift to shorter wavelengths. This can be distinguished from that of the substrate, because of the reduction of the eximer form inside the membrane (EM: 470nm @ EX: 390nm). This assay is quantitative and can be used with mammalian or bacterial cell lines. It can also be used in a purified enzyme format with the addition of lipid vesicles or added membrane components. This assay has also found use in measurement of endothelial lipases (EL) and lipoprotein lipase (LPL). For more information about this assay see the references below: NOTE: Marker Gene also sells a convenient Fluorescent Lipase Assay Kit (M0612) with all of the reagents and a detailed protocol for measuring lipase activity in living cells. Dousset, N., Negre, A., Salvayre, R., Rogalle, P., Dang, Q.Q., Douste-Blazy, L. (1988). “Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase.” Lipids 23: 605-608. Main, L.A., Okumura-Noji, K., Ohnishi, T., Yokoyama, S., (1998) “Cholesteryl ester transfer protein reaction between plasma er R., (2000) “Methods for lipase detection and assay: a critical review” Eur. J. Lipid Sci. Technol.2000: 133–153. lipoproteins.” J. Biochem. (Tokyo) 124: 237-243. Negre-Salvayre, A., Abouakil, N., Lombardo, D., Salvayre, R., (1990) "Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase." Lipids 25(8): 428-434. Beisson F., Tiss, A., Rivière C., Verg
	Stock up and Save! Marker Gene will introduce a price increase of 5% on most products and kits from our catalog, starting on January 1^st, 2006. Orders placed and received before January 1^st will still be credited for our 2005 prices. So place your orders now, online, through one of our distribution partners, by FAX (1-541-342-1960) or by telephone (toll-free) at 1-888-218-4062, and save. For more information on the new 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.
	New lacZco Vector Systems. Marker Gene offers a variety of new vectors with improved expression profiles for the lacZ (b-galactosidase) gene (M1015, pCMVblacZco; M1016, pSV40blacZco, M1017 pCMVblac Znls12co and M1018 pSV40blac Znls12co). These vectors contain a coding sequence for the E. coli lacZ gene that has been codon-optimised when expressed in mammalian cells. This results in the expression of b-galactosidase at levels up to 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of b-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. The nls12 sequence variant also targets the protein for nuclear localization by the addition of a twelve amino acid sequence, ProLysLysLysArgLysValGluAspProLysAsp (from the SV40 T antigen nuclear localization signal) after the methionine initiation residue. Marker Gene is currently working with Dr. Donald Anson at the Department of Chemical Pathology, Women's and Children's Hospital in North Adelaide, Australia to also provide newly developed lacZ expression lentiviral vectors with high expression efficiency in mammalian cells. When used in such a lentiviral construct, the codon-optimised gene gives an approximately five-fold increase in apparent titre, as determined by X-Gal staining, in comparison to an analogous construct containing the native E. coli gene. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised b-galactosidase gene has a number of advantages over the native E. coli gene sequence. For more information on these new vector systems, see the references below or visit our website. Look for these new vectors in kits from Marker Gene soon! Anson D.S., Limberis M., (2004) “An improved beta-galactosidase reporter gene.” J Biotechnol 108(1): 17-30. http://biology.kenyon.edu/courses/biol09/betagal/FRAMES/ MacGregor, G. R. & Caskey, C. T. (1989) “Construction of plasmids that express E. coli beta-galactosidase in mammalian cells.” Nucleic Acids Res. 17:2365. Okayama, H. & Berg, P. (1983) “A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.” Mol. Cell Biol. 3:280-289. MacGregor, G. R.,et al. (1987) “Histochemical staining of clonal mammalian cell lines expressing E. coli beta-galactosidase indicates heterogeneous expression of the bacterial gene.” Somat. Cell Mol. Genet. 13:253-265.
	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.