Marker Gene Monthly Newsletter   

May, 2005

Volume 5, Number 5

© 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.

Embryonic Stem Cell Gene Trap Methods.

The gene trap strategy is an insertional mutagenesis technique involving integration of an exogenous DNA sequence, termed a trap vector, as a mutagen that produces a stable mutation in the host genome.  The trap vectors are typically promoterless, and rely on insertion into an active gene (via intron splicing) for expression.  They also contain a marker gene sequence that facilitates the identification and isolation of the mutated gene sequence. The trap vector’s expression therefore mimics that of the mutated endogenous gene.  Simple staining for the marker gene (typically using lacZ substrates like FDG (M0250) or X-Gal reveal intricate expression patterns for the trap vector and allow the researcher to select for cell lines, tissues or whole animals that contain the expression patterns they desire.  The trap vector also often contains a selection marker (neo) that allows sorting of cells that incorporate an active inserted vector.  Gene trap is an increasingly powerful method for identifying genes important in biological phenomena.  Several databases have been established, including those by the Sanger Institute (http://www.sanger.ac.uk/PostGenomics/genetrap/), the University of Manitoba (http://www.igtc.ca/) and the German GeneTrap consortium (http://tikus.gsf.de/) to act as a repository of the over 20,000 existing gene trap sequences thusfar identified.  Moreover, the method has been used to produce mutant organisms whose phenotypes provide invaluable information about the biological functions of the genes responsible for these phenotypes. Indeed, a number of genes essential for mouse embryogenesis have been identified by the gene trap method.  For example, the lacZ positive mouse TgR(ROSA26)26Sor, where lacZ is expressed in all tissues and organs, has been developed using this method.   For more information on gene trap techniques, and the powerful methods of analysis they provide, please see the references below or visit our website.

• Skarnes W.C., Auerbach B.A., Joyner A.L., (1992) “A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice.” Genes Dev. 6(6): 903-18.
• Kawaguchi J., Wilson V., Mee P.J., (2002) “Visualization of whole-mount skeletal expression patterns of LacZ reporters using a tissue clearing protocol.´ Biotechniques 32(1): 66, 68-70, 72-3.
• Paola Menichini, Silvia Viaggi, Elena Gallerani, Gilberto Fronza, Laura Ottaggio, Alberto Comes, Joachim W. Ellwart, Angelo Abbondandolo 1,3 (1997) “A gene trap approach to isolate mammalian genes involved in the cellular response to genotoxic stress” Nucleic Acids Research  25(23):  4803–4807.
• Sutherland, H.G.E., Mumford, G.K., Newton, K., Ford, L.V., Farrall, R., Dellaire, G., Caceres, J.F., Bickmore, W.A. (2001) “Large-scale identification of mammalian proteins localized to nuclear sub-compartments.” Human Mol. Gen. 10(18): 1995-2011.
• Gossler, A., Joyner, A.L., Rossant, J., Skarnes, W.C. (1989) “Mouse Embryonic Stem Cells and Reporter Constructs to Detect Developmentally Regulated Genes” Science 244(4903): 463-465.
• Zambrowicz, B.P., Imamoto, A., Fiering, S., Herzenberg, L.A., Kerr, W.G., Soriano, P., “Disruption of overlapping transcripts in the ROSA bgeo 26 gene trap strain leads to widespread expression of b-galactosidase in mouse embryos and hematopoietic cells” Proc. Natl. Acad. Sci. USA 94: 3789-3794.
•   Reddy S., Rayburn H., von Melchner H., Ruley, H.E., (1992) “Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.” Proc. Natl. Acad. Sci. USA 89(15): 6721-5.
  

Mammalian GUS Marker Gene Systems.

The use of the Esherichia coli uidA (GUS) gene as a marker gene in mammalian cells has been widely overlooked.  Several recent reports, however, point to the use of GUS as a alternative to other reporters, especially when multicolor analysis is required.  GUS has several advantages for use, including the many substrates that are available with various colors (X-GlcU, Red-GlcU) or fluorescence (4-Methylumbelliferyl-b-D-Glucuronide (MUGlcU), (360/449) M0240; Carboxyumbelliferyl-b-D-Glucuronide (CUGlcU) M0256; Fluorescein di-b-D-Glucuronide, di-methyl ester (490/520)   M0969) as well as chemiluminescent emission M0856, allowing a range of assays.  Cells expressing the GUS enzyme can be analyzed in mixed cell populations, and live cells can be separated and isolated according to their GUS activity by FACS analysis.  GUS activity can be histochemically monitored at the single-cell level, and GUS protein can be quantitated by mRNA analysis or by using a highly sensitive fluorescent or chemiluminescent enzyme assays. Although low level b-glucuronidase activity is found in mammalian cells, these endogenous GUS enzymes have a pH optima in the acidic range (3.8 to 5.0).  The recombinant GUS enzyme from E. coli has a pH optimum of 7.4.  This permits assay in the presence of the low-level endogenous GUS activity at a neutral pH that is optimal for the bacterial but not for the mammalian enzyme.  Finally, the GUS coding sequence is relatively small (1.8 kb), allowing a total of 2.5 kb of exogenous sequences to be cloned into expression vectors.  For more information about GUS assays and techniques, please visit our website or see the references below. 

• Gallie, D R.; Walbot, V.; Feder, J N. GUS as a useful reporter gene in animal cells. In: Gallagher S R. , editor; Gallagher S R. , editor. GUS protocols. San Diego, Calif: Academic Press, Inc.; 1992. p. 181–188.
•  Lorincz M; Roederer M; Diwu Z, Herzenberg, L.A., Nolan, G.P., (1996) “Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase.”  Cytometry  24(4): 321-9.
•  Albert Spicher, Oivin M. Guicherit, Laurent Duret,  Aaron Aslanian, Elvira M. Sanjines, Nicholas C. Denko,  Amato J. Giaccia,  and Helen M. Blau (1998) “Highly Conserved RNA Sequences That Are Sensors of Environmental Stress” Mol Cell Biol.  18(12): 7371–7382.
• DeWet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R., Subramani, S. (1987) “Firefly Luciferase gene: Structure and expression in mammalian cells.”  Mole. Cell Biol. 7: 725-737.

Mechanism of Luciferase Action.

Firefly luciferase has presented itself as a nearly ideal reporter gene for plant and animal cells.  Although there are now several luciferases available, the first luciferase gene cloned after isolation was from the North American firefly Photinus pyralis.  The native gene contains several introns, but the full-length cDNA also has been isolated, and several important modifications have been made to current cloning vectors for stability and analysis. The gene codes for an active enzyme that is a single polypeptide with a mass of 62kD.  The mechanism of the luciferase reaction results in the emission of a yellow-green light (565 nm) and requires only the enzyme, ATP, Mg²⁺, O₂ and the substrate D-luciferin (M0237, M0626) for activity.  In fireflies, the luciferase reaction occurs in the peroxisomes of a specialized light organ (lantern), but after cloning, the reaction can also occur in many cell types including plants, mammalian cells, in bacteria and in cell-free extracts. The only equipment required to detect luciferase activity is a photon-measuring device such as a phototube luminometer, a scintillation counter or even photographic film.

The enzyme firefly luciferase (Photinus-luciferin:oxygen 4-oxoreductase [decaboxylating, ATP-hydrolysing] (EC 1.13.12.7) produces light by the ATP-dependent oxidation of D-luciferin.  In the presence of D-luciferin and ATP, an enzyme-bound luciferyladenylate complex is formed and this is followed by oxidative decarboxylation with the production of CO₂, oxyluciferin, AMP and light.  The chemical energy of the in vitro reaction can proceed with a very high quantum yield (0.88 at pH 7.9).  This implies that several of the steps to produce the excited state intermediates in this process occur with high efficiency.  Current knowledge indicates that in the first step, the enzyme luciferase binds D-luciferin, adenosine triphosphate (ATP) and Mg⁺² to produce a mixed anhydride on the 4-carboxyl group releasing pyrophosphate (PPi).  This can be a reversible reaction, but since ATP is consumed, the equilibrium lies far to the right.  The resulting luciferyl-AMP derivative (adenyl-luciferin) is now itself a substrate for luciferase as a highly activated intermediate which can be acted upon by molecular oxygen.  Oxygen adds at the 4-position of adenyl-luciferin and cyclization to a dioxetane intermediate occurs spontaneously, with the loss of AMP.  A rearrangement of this dioxetane produces the overall oxidation and decarboxylation of the parent D-luciferin, and the excited-state keto-intermediate, oxyluciferin is produced.  Using labeled oxygen, ¹⁸O₂ , it has been found that the carbon dioxide released contains only one ¹⁸O atom, and  the other ¹⁸O oxygen adds to the C-4 keto group. 

This excited-state oxyluciferin, not the dioxetane intermediate, is the proposed light emitting compound.  Excited oxyluciferin can decompose in two ways, giving two different colors of emission.  At lower pH’s (pH of about 6) a red light emission is possible (EM apporx. 630 nm).  However, in typical biochemical systems, oxyluciferin will decompose to give the normal yellow-green light of about 565 nm for enzyme reactions run at or near pH 8.  Nevertheless this pH dependence can be important for in vitro assays, and since common phototubes have greater sensitivity in the blue rather than red spectral regions, shifts to longer wavelengths will give apparently lower levels of activity.  Finally the presence of urea, divalent zinc, cadmium or mercury ions can cause a similar longer wavelength shift in emission.  For more information about the luciferase reaction and assays, please see our website or the references below. 

• Branchini B.R., Southworth T.L., Murtiashaw M.H., Magyar R.A., Gonzalez S.A., Ruggiero M.C., Stroh J.G.,  (2004) “An alternative mechanism of bioluminescence color determination in firefly luciferase.” Biochemistry 43(23): 7255-62.
• Wannlund J., DeLuca M., Stempel K., Boyer P.D., (1978) “Use of 14C-carboxyl-luciferin in determining the mechanism of the firefly luciferase catalyzed reactions.” Biochem. Biophys. Res. Commun. 81(3): 987-92.
• Tsuji F.I., DeLuca M., Boyer P.D., Endo S., Akutagawa M., (1977) “Mechanism of the enzyme--catalyzed oxidation of Cypridina and firefly luciferins studied by means of 17O2 and H218O1.” Biochem Biophys Res Commun 74(2): 606-13.
• Ow DW, et al. (1986) "Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants." Science 234: 856.
• Gildea J.J., Harding M.A., Gulding K.M., Theodorescu D., (2000) "Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement." Biotechniques 29(1): 81-86.
•   Van Leeuwen W., et al. "The use of the luciferase reporter system for in planta gene expression studies." (2000) Plant Mol. Biol. Rep. 18: 143a
• Matthews B.F., Saunders J.A., Gebhardt J.S., Lin J.J. Koehler S.M., (1995) "Reporter genes and transient assays for plants." Methods Mol. Biol. 55: 147-162.

 Chloramphenicol Acetyl Transferase (CAT) Assay Systems. 

A very popular reporter gene is chloramphenicol acetyltransferase or CAT.  The CAT bacterial gene evolved to neutralize the antibiotic chloramphenicol.   The CAT enzyme transfers acetyl or other acyl groups from CoA to hydroxyls on chloramphenicol. Molecular biologists were quick to recognize this enzyme as a potential reporter protein in eukaryotic cells since it is not present in these cell lines.  To determine expression levels, one can either measure the mRNA or protein production. Measurement of mRNA is performed by in situ hybridization assay. But more popular enzyme assays have been developed, by extracting the proteins from cells and then mixing this extraction solution with chloramphenicol and actyl-CoA that has been synthesized with radioactive forms of hydrogen (³H) or ¹⁴C. In addition, the substrate acetyl CoA is also sometimes prepared in-situ during the assay (using Acetyl CoA , ATP, Acetyl CoA Synthetase and [³H]-Acetic Acid) and added to the mixture. The amount of acetylation is directly proportional to the amount of CAT enzyme present. Therefore, one can measure the amount of acetylated chloramphenicol in different protein extractions and determine how much protein was produced as a result of activate promoters, for example.  CAT can add two acetyl groups to each chloramphenicol, but the reaction will usually produce a mixture of mono and di-acetylated products.

When the CAT reactions is completed, the products are typically placed on thin-layer chromatographic (TLC) sheets, placed in the appropriate solvent (a mixture of chloroform and methanol) and allowed to migrate up the TLC sheet.  When the TLC sheet is dried, it is exposed to X-ray film (see above).  Each product (two forms of chloramphenicol with one acetyl group and one with two acetyl groups added) and all the unused substrate (acetyl CoA, chloramphenicol and CAT) will migrate on the TLC surface according to their ability to interact with surface of the TLC. If ¹⁴C-labeled chloramphenicol is used, the all radioactive molecules containing chloramphenicol will be visible on the X-ray film.  If ³H-acetyl-CoA is used, chloramphenicol and all its acetylated forms will be visible (Lane 4).  Newer assays take advantage of butyrl-CoA activity and organic solvent extraction.  For more information about these assays and methods, please see our website or the references below.

• Seed, B. and Sheen, J.-Y. (1988) “A simple phase-extraction assay for chloramphenicol acyltransferase activity. “ Gene 67: 271-277.  
• Sleigh, M., (1986) “A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal. Biochem. 156: 251-256.
• Tomizawa, J.-i., (1985) “Control of ColEl plasmid replication: initial interaction of RNA1 and the primer transcript is reversible.” Cell 40: 527-535.
• Wu Y., Sifri C.D., Lei H.H., Su X.Z., Wellems T.E., (1995) “Transfection of Plasmodium falciparum within human red blood cells.” Proc. Natl. Acad. Sci. USA 92(4): 973–7.
• Sankaran L. (1992) “A simple quantitative assay for chloramphenicol acetyl-transferase by direct extraction of the labeled product into scintillation cocktail.” Anal Biochem 200(1):180–6.
• Lucas, S.J., Holder, A.H., (2004) “An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection” Molecular & Biochemical Parasitology 136: 287–296
  
  

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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
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• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
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