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Marker Gene Technologies, Inc
MGT is a primary manufacturer. Order from the source! Kits for Marker Genes Detection, Fluorescent Reagents, Labelling Reagents & Cell Regulatory Substrates.

Removal of Marker Genes.

Marker genes find numerous applications in animal and plant systems, but can also cause consumer concern when used in commercial products or add regulatory requirements from the presence of "excess" exogenous DNA.  Several methods have recently been introduced to remove marker genes in plants, using site-specific recombination systems (e.g., lox/Cre recombinase, Flp recombination target (FRT)/Flp recombinase, or Rs/R recombinase) and by using an inducible promoter system (e.g. for b-estradiol) for the recombinase gene.  In these recombination schemes, the marker gene is flanked by recombination sites, such as lox, FRT, or Rs, which specifically interact with a recombinase protein (e.g., Cre, Flp, or R, respectively).  This interaction promotes recombination between the sites and deletes the marker DNA from the host genome.  For more information about these techniques see the references below.

• David W. Ow The right chemistry for Marker Gene removal? (2001) Nature Biotechnol. 19(2): 115  116.

• Zuo, J., Niu, Q.-W., Moller, S.G. & Chua, N.-H. Chemical-regulated, site-specific DNA excision in transgenic plants. Nat. Biotechnol. 19, 157-161 (2001).

• Sugita, K., Kasahara, T., Matsunaga, E. & Ebinuma. H. A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency Plant J. 22, 461-469 (2000). 

Small Molecule Activator of Caspase-3.

Professor Xiaodong Wang, and co-workers at the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX have identified a small molecule activator of apoptosis, -(trichloromethyl)-4-pyridineethanol (PETCM), using high-throughput screening in a panel of cancer cells in culture.  PETCM was found to be a selective activator of caspase-3.  Caspase-3 is an important member of the family of enzymes implicated in the initial events of programmed cell death.  The identification of these types of active compounds, point the way toward understanding the death initiator processes in tumor cells (two proteins were implicated in the present study), and may lead to new types of chemotherapies for intractable cancers.  For more information about this work, see the references below.

• Distinctive Roles of PHAP Proteins and Prothymosin-a in a Death Regulatory Pathway, X. Jiang, H.E. Kim, H. Shu, Y. Zhao, H. Zhang, J. Kofron, J. Donnelly, D. Burns, S.C. Ng, S. Rosenberg, X. Wang, Science 299 (2003) 223-226.

• Apoptosis: Life and Death Decisions D.W. Nicholson and N.A. Thornberry, Science 299 (2003) 214-215.

Quantum Dot Fluorescence Applications.

Quantum dots (Qdots) are polymer-coated beads that contain nanometer scale microcrystals of semiconductor material (cadmium selenide).  Arising from the solar energy research (photovoltaic cell conversion studies) of the 1980s, they can be excited at a wide range of wavelengths (300-500nm) and produce bright fluorescence emission, with the color dependent upon the size of the contained nanocrystals.  In addition, they are much more photostable than typical fluorescent dyes.  Many quantum-dot bio-conjugates have been prepared and used in cell labeling studies, and even intracellularly, with uptake by endocytosis.  For more information, see the references below.

• Rosenthal, S.J., Tomlinson, I., Adkins, E.M., Schroeter, S., Adams, S., Swafford, L., McBride, J., Wang, Y., DeFelice, I.J., Blakely, R.D., Targeting Cell Surface Receptors with Ligand Conjugated Nanocrystals. J. Am. Chem. Soc. 124(17):4586-4594 (2002).

• kerman, M.E., Chan, W.C., Laakkonen, P., Bhatia, S.N., Ruoslahti, E., Nanocrystal Targeting In Vivo. Proc. Natl Acad. Sci. USA 99:12617-12621 (2002).

• Watson, A., Xingyong, W., Bruchez, M., Lighting Up Cells with Quantum Dots. (2003) Biotechniques 34(2): 296-303.

Stems Cells found Totipotent by lacZ Staining.

Dr. Diana Clark and colleagues at the Medical Nobel Institute in Stockholm, Sweden traced the differentiation of neural stem cells by lacZ transfection and staining.  They found these cells could be induced to differentiate into most of the cells of the body.  This work is promising for use of stem cells to potentially re-populate damaged tissue after stroke or spinal cord injury or aide in regenerating tissues in multiple sclerosis, diabetes or neurodegenerative diseases.  For more information about these studies, see the references below.  Marker Gene sells several products for sensitive and specific staining of lacZ-positive cells and tissues (including: M0250; M0259; M0241; M0255; and M0257).

• Clarke, D. L. Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U, Frisen J., Generalized potential of adult neural stem cells. Science 288, 1660-1663 (2000).

• McKay, R., Mammalian deconstruction for stem cell reconstruction Nature Medicine (2000) 6( 7):747-748.

a- Galactosidase Marker Gene for Yeast.

The yeast two-hybrid screen is a genetic method of detecting protein-protein interactions in vivo.  Positive clones are selected by their ability to activate the transcription of a reporter gene, which also enables them to grow on nutritionally selective media.  Most two-hybrid methods use the E. coli lacZ gene as the reporter gene.  Usually the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ by a filter-lift assay.  Marker Gene currently produces several substrates that are useful for b-Gal detection in yeast strains (M0250, FDG; M0203, Resorufin-Gal; M0252, TFMU-Gal, M0257, CUG).   Recently yeast strains producing the a-galactosidase marker gene have been developed and used to assay GAL4-based two-hybrid interactions directly on nutritional selection plates with the chromogenic substrate X-a-Gal.  Look for new products from Marker Gene in this area in the near future.  For more information about these techniques, see the references below.

• S. Aho, A. Arffman, T.Pummi and J. Uitto, A novel reporter gene MEL1 for the yeast two-hybrid system. Anal. Biochem., 253, 270-272  (1997).

• P. Chevalier, D. Roy and and L. Savoie, X-a-gal-based medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk., J.Microbiol. Meth., 13, 75 (1991).

• R. Gossrau and Z. Lojda, Histochemical detection of a-D-galactosidase with 5-Br-4-Cl-3-indoxyl a-D-galactoside. Acta Histochem., 85, 213 (1989).

• R.S. Tubb and P.L. Liljestrom, A colony-colour method which differentiates a-galactosidase positive strains of yeast. J. Inst. Brew. 92, 588, (1986).

Toward a Marker Gene for Colorectal Tumors.

The long-standing hope of finding specific reporter genes that are upregulated in tumors has recently shown renewed promise in work published by Dr. Andrew Feinberg and his group at Johns Hopkins University School of Medicine.  By examining the DNA methylation patterns of patients with genetic predisposition to colorectal cancer or who have been diagnosed with the disease, they found statistically higher levels of gene silencing in the gene IGF2 (insulin-like growth factor 2) in these patients (5X or 21X higher respectively).  These assays were performed using a reverse transcription of mRNA isolated by biopsy from colonoscopy tissue samples, using a PCR format with specific primers.  The levels of methylation were analyzed by a bisulfite genomic sequencing method.  For more information about these techniques, please see the references below.

• H. Cui, M. Cruz-Correa, F. M. Giardiello, D. F. Hutcheon, D. R. Kafonek, S. Brandenburg, Y. Wu, X. He, N. R. Powe, and A. P. Feinberg, Loss of IGF2 Imprinting: A Potential Marker of Colorectal Cancer Risk Science 299 (2003) 1753-55.

• H. Uejima, M. P. Lee, H. Cui, and A. P. Feinberg, Nature Genet. 25, 375-376 (2000).

• Feinberg, AP: Genomic imprinting and cancer. In The Metabolic and Molecular Bases of Inherited Disease, 8th ed. Scriver CR, Beaudet AR, Sly W, Valle D (eds), McGraw-Hill, New York, pp 525-537, 2001.

Hydrophobic Stains for 2D-SDS-PAGE.

The ability to stain proteins inside SDS-PAGE gels offers a significant advantage over chemical labeling techniques or blotting / post-electrophoresis staining and destaining methods.  John Shultz and Gregg Larson (Promega Corp., Madison, WI) recently introduced a number of hydrophobic analogs of fluorescent dyes (fluorescein, Dansyl chloride, carbocyanines, etc.) that can be used for direct and quantitative labeling of proteins inside polyacrylamide gels.  The stains bind to the SDS hydrophobic protein coat and exhibit low background fluorescence.  For more information about these stains and techniques, please see the references below.

• Larson, Gregg A.; Shultz, John W., Applications Of The Chromaphor Protein Recovery System. BioTechniques, 15:316-323.

• Shultz, John W., Larson, Gregg A.., Protein Staining Compositions and Methods, US Patent 5,705,649 (1998).

• Kendrick, N., Laser scanning quantification of 2-D gel spots using ChromaPhor^TM Green Stain, Promega Notes Magazine 37 (1992) 11.

AHAS Reporter Gene in Plants.

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) also known as acetolactate synthase, is an herbicide-resistance selection marker used in plant cell studies.  It catalyzes the first step in branched-chain amino-acid (valine, leucine and isoleucine) biosynthesis, and when a modified version is cloned into plants, detoxifies the inhibition of the enzyme by sulfonylurea herbicides slufometuron methyl and chlorosuluron (marketed by Du Pont as the active ingredients in Oust and Glean, respectively) and the inidazolinone herbicides 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-nicotinic acid and 2-(4-isopropyl-4-methyl-5-oxo-2-inidazolin-2-yl)-3-quinolinecarboxylic acid (marketed by American Cyanamid as the active ingredients in Arsenal and Scepter, respectively).  The recombinant plants carry a mutation (changing the serine (coded by AGT) at position 621 to asparagine (AAT), which provides a relief of the inhibition by these herbicides.  An assay of this AHAS reporter has been developed, involving conversion of the enzymatic product acetolactate to acetoin, followed by detection as a complex with creatine and naphthol.  For more information about this selection marker gene in plants see the references below.

• Singh, B.K., Stidham, M.A., Shaner, D.L., Assay of Acetohydroxyacid Synthase Anal. Biochem.  171 (1988) 173-179.

• Hill, C.M., Pang, S.S., Duggleby, R.G., Purification of E.Coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. Biochem. J. 327 (1997) 891-898.

• Schloss, J.V., Van Dyk, D.E., Vasta,. J.F., Kutny, R.M., Purification and Properties of Salmonella typhimurium Acetolactate Synthase Isozyme II from E. Coli HB101/pDU9, Biochemistry 24 (1985) 4952-4959.

• Zhu T, Peterson DJ, Tagliani L, St. Clair G, Baszczynski CL, and Bowen B. 1999. Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proceedings of the National Academy of Science 96:8768-8773.

• Zhu T, Mettenburg K, Peterson DJ, Tagliani L, and Baszczynski CL. 2000. Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Nature Biotechnology 18:555-558.

Bacterial Detection using FDG

Fluorescein di-b-D-Galactopyranoside (FDG, M0250) is a popular substrate for measuring cloned beta-Galactosidase activity in living cells.  It has also found utility in staining and quantifying bacteria, as well as in isolating specific strains of bacteria by fluorescence activated cell-sorting (FACS) analysis.  Interestingly, FDG was found to be about 70 times more sensitive in bacterial  assays than GFP.  For more information about FDG staining of bacteria, see the references below:

• b-Galactosidase Activity in Single Differentiating Bacterial Cells." F. Russo-Marie, Roederer, M. Sager, B., Herzenberg, L., Proc. Natl. Acad. Sci. USA 90:8194 (1993).

• Nelis, H.; Van Poucke, S. Enzymatic detection of coliforms and Escherichia coli within 4 hours." Water, Air, and Soil Pollution (2000), 123(1-4): 43-52.

• Dreier, Jurg; Breitmaier, Eva B.; Gocke, Elmar; Apfel, Christian M.; Page, Malcolm G. P. Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay." Mutation Research (2002), 513(1-2): 169-182.

• Rowland B ; Purkayastha A ; Monserrat C ; Casart Y ; Takiff H ; McDonough KA (1999) Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species FEMS Microbiol. Lett. 179(2): 317-25

Jet-Lag and Circadian Rhythms in Plants.

The effects of daylight length and time-changes, and the physiological events that occur in eukaryotic cells in response to these events, have long intrigued scientists.  Some of these processes are beginning to be understood in plants and animals by cloning marker genes (for example firefly luciferase, luc) into cells under control of specific circadian promoter elements.  By adding the reagent D-luciferin (M0237) to these cloned tissues, Steven Kays lab at the Scripps Research Institute in San Diego (see: http://www.scripps.edu/cb/kay/) has identified several new genes that light up, i.e. are responsible for the physiological changes in plants and animals, when daylight and night-time cycled gene expression occurs.  For more information, see the references below:

• Covington, M.F., Panda, S., Liu, X.L., Strayer, C.A., Kay, S.A., Wagner, D.R. (2001) ELF3 modulates resetting of the circadian clock in Arabidopsis. The Plant Cell 13, 1305-1316.

• Millar A.J. and Kay S.A. (1996). Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis. Proc. Natl. Acad. Sci. USA, 93, 15491-15496.

• Millar A.J., Straume M., Chory J., Chua N.-H. and Kay S.A. (1995) The regulation of circadian period by phototransduction pathways in Arabidopsis. Science, 267, 1163-1166.