Zinc Finger Nuclease Proteins. 
Zinc finger proteins are derived from transcription factor proteins and are designed to bind to specific DNA sequences. By combining zinc-finger proteins with endonucleases, Zinc-Finger Nucleases (ZFNs) can be produced. These ZFNs can then bind to and cut doubly stranded DNA in chromosomes at specific sites. Upon double-stranded DNA cleavage, a foreign segment of DNA can then be inserted at the strand breakage site by a process similar to homologous recombination. Because zinc-finger nucleases bind to specific 3 base-pair sites, the position of the strand breakage can be tailored to insert a foreign gene construct at a specific position in the chromosome, thereby leading to a stably transformed cell. At present, three and four-finger ZFNs, with each zinc finger protein binding to a 3-base codon, have been designed to recognize up to 12-bp regions, and with the addition of other DNA binding domains from transcription factors or repressors, a tailored binding domain can be constructed for insertion into a selected target sequence. These methods have also been used to produce selective knock-outs of particular genes in vivo.
To date, ZFNs that can bind to any of the 64 possible three-base combinations have not yet been developed. Nonetheless, a three finger ZFN has been used to correct a defective GFP marker gene in mammalian cells. And a designed four-finger ZFN that recognized an endogenous target site within the IL2Rγ gene that causes the human disease Severe Combined Immune Deficiency (SCID) was able to modify diseased cells highly efficiently and permanently to correct the mutation of the defective IL2Rγ gene. Although the current levels of insertion are usually low, these methods promise to have a significant impact on the ability to modify genes within the chromosomes of living cells. To find out more about these methods, please see the references below or visit our website.
- Gommans WM, Haisma HJ, Rots MG, (2005). "Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command." J.Mol. Biol. 354(3): 507-519.
- Durai S, Mani M, Kandavelou K, Wu J, Porteus M, Chandrasegaran S (2005). "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Res 33(18): 5978-5990.
- Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) "Highly efficient endogenous human gene correction using designed zinc-finger nucleases." Nature 435646–435651.
- Kaiser J (2005) "Putting the Fingers On Gene Repair." Science 310(5756): 1894-1896.
- Porteus MH, Baltimore D (2003) "Chimeric Nucleases Stimulate
Gene Targeting in Human Cells." Science 300
- Photo: http://www.laboratorytalk.com/news/sgm/sgm210.html
In vivo Chemiluminescent b-Gal Assays.
 b-Galactosidase (lacZ) and firefly luciferase (luc) are two of the most widely used marker genes for measuring gene expression levels, for promoter activity analysis, to measure protein levels or mobility through fusion proteins, or for use in protein-protein interaction studies based on the yeast two-hybrid enzyme complementation assays, as well as many other applications. Recently, the combined ultrasensitive detection substrate, D-Luciferin-6-O-b-D-Galactopyranoside (M1087) (also termed Beta-Glo®) has been used for in vivo analysis of lacZ beta-galactosidase activity in a variety of systems. This substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-galactosidase before it can be catalyzed by firefly luciferase (luc) to generate light. As a result, bioluminescence becomes dependent on lacZ expression and activity. Using this substrate, methods have been developed to monitor expressed beta-galactosidase levels in transgenic lacZ cell lines as well as inducible tissue-specific lacZ expression in vivo, noninvasively and without incident light sources for illumination. Another advantage of using lacZ beta-galactosidase as a bioluminescent probe is that this enzyme does not require ATP or other cofactors for extracellular enzyme detection methods, in contrast to luciferase which requires such intracellular cofactors. As a result, antibodies conjugated to the beta-galactosidase enzyme can be used to detect specific cells or tissues through extracellular antigens in vivo. Coupling of the ultrasensitive chemiluminescent detection properties of firefly luciferase system to the advantages of lacZ beta-galactosidase permits bioluminescent imaging applications that were previously not obtainable by other means.
Recent work from the Laboratory of Dr. Helen Blau and coworkers at the Baxter Laboratory for Stem Cell Biology, Stanford University have identified D-luciferin-6-O-b-D-galactopyranoside (M1087) as a potential new probe for measurement of lacZ activity in vivo. Transgenic cells carrying the lacZ gene, either injected into the muscle or subcutaneously in nude mice or from myf-5-lacZ positive mice, were treated with D-luciferin-6-O-b-D-galactopyranoside (M1087) intraperitoneally and imaged using a high-resolution CCD camera. The location of luminescence was evident in the transgenic cells or tissues upon long duration exposures (up to 180 sec.). Although the probe was found to be unstable when injected intravenously, the D-luciferin-6-O-b-D-galactopyranoside (M1087) substrate was found to be non-toxic under these conditions. These results demonstrate that transgenic b-Gal activity can be monitored by using sensitive bioluminescence detection. The implications for use of these techniques to monitor other enzyme activities in vivo are evident. To find out more about these methods, please see the references below or visit our website.
- von Degenfeld G, Wehrman TS, and Blau HM (2009) "Imaging β-Galactosidase Activity In Vivo Using Sequential
Reporter-Enzyme Luminescence." Methods Mol Biol. 574: 249–259.
- Geiger R, Schneider E, Wallenfels K, Miska W, (1992) “A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase” Biol. Chem. Hoppe-Seyler 373(12): 1187-1191.
- Ugarova NN, Voznyi Y, Ya V, Kutuzova GD, Dement'eva E I (1991) “Bioluminescent assay of b-galactosidase using D-luciferin-o-b-galactoside.” Biolumin. Chemilumin. Proc. Int. Symp., 6th (1991), Meeting Date 1990, pp. 511-14. Publisher: Wiley Intersceince, Chichester, UK Editor(s): Stanley, Philip E., Kricka, Larry J.
- Wehrman TS , von Degenfeld G , Krutzik PO , Nolan GP , Blau HM (2006) “Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence.” Nature methods 3(4): 295-301.
- Geiger R , Schneider E, Wallenfels K, Miska W (1992) “A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase.” Biological chemistry Hoppe-Seyler 373(12): 1187-91.
Live Cell Luciferase Assays. 
Expression levels of the firefly luciferase (luc) marker gene are typically measured using a cell lysis methodology (see our MarkerGene™ Live Cell Luciferase Assay Kit; M0626 for a standard protocol) by adding D-luciferin and ATP containing buffers. But often it can be of interest to visualize the bioluminescent signal directly in cells grown in culture. Although the signal obtained using this method is not quantitative and of low light, visualizing luciferase activity in live cell culture has certain advantages including measurement of the percent transfection, and also for estimation of qualitative levels of transfection between different cell samples or transfection methods. Direct imaging can be accomplished by the use of digital microscopy using high-resolution CCD camera analysis with cell permeable luciferin analogs like D-Luciferin, ethyl ester (M0906). Cells are typically bathed in a solution containing
500 mM of the membrane
permeable luciferin substrate (M0906),
530 mM ATP, 20 mM Tricine, 2.7 mM MgSO4 ,
0.1 mM EDTA and 33.3 mM DTT in 500 mL DMEM, without FCS for 10 min., followed by image collection using a 5 min. integration time. The ethyl ester of the D-luciferin ethyl ester is removed by intracellular esterase activity to provide the reactive D-luciferin substrate intracellularly. Comparison of light emitting cell vs. Nomarski (total) cell counts will give an estimate of percentage transfection. For more information about these methods, please see the references below, or visit our website.
- Kratzer S, Mundigl O, Dicker F, Seeber S, (2001) "Digital imaging microscopy of firefly luciferase activity to directly monitor differences in cell transduction efficiencies between AdCMVLuc and Ad5LucRGD vectors having different cell binding properties." J. Virol. Meth. 93(1-2): 175-179.
- Mettenleiter TC, Grawe W, (1996) "Video imaging of firefly
luciferase activity to identify and monitor herpesvirus infection
in cell culture." J. Virol. Meth. 59: 155–160.
- Craig FF, Simmonds AC, Watmore D, McCapra F,
White MRH, (1991) "Membrane-permeable luciferin es-ters
for assay of firefly luciferase in live intact cells."
Biochem. J. 276: 637–641.
Long Wavelength D-Luciferin Analogs.
 The bioluminescent reaction of D-luciferin with its enzyme, firefly luciferase, procedes through a high-energy oxyluciferin intermediate. Stabilization of the keto versus enol forms of this intermediate can cause a long-wavelength shift of light emission. Recently, several groups has demonstrated that modifications of the D-luciferin structure can enhance the stability of the oxyluciferin intermediates and cause even further red shifts in the emission spectrum for the luciferin-luciferase reaction. Most of these modifications have been to the benthiazole ring. For example, by using the cyclic aminoalkyl, 6-aminomethyl or 6,6-dimethylamino derivatives of aminoluciferin as substrates, the bioluminescent emission for the reaction with firefly luciferin can be red shifted out to ~623 nm. In addition by using the 5,5-dimethyl-D-luciferin derivative, similar red shifts can be obtained. The possibility to utilize these new substrates for in vivo applications of luciferase marker genes in living tissues is significant. For more information about these assays, please see the references below or visit our website.
- Reddy GR, Thompson WC, Miller SC (2010) "Robust Light Emission from Cyclic Alkylaminoluciferin Substrates for Firefly Luciferase." J. Amer. Chem. Soc. 132: 13586-13587.
- Branchini BR, (2000) "Chemical synthesis of firefly luciferin analogs and inhibitors." Methods Enzymol. 305: 188–195.
- Woodroofe CC, Shultz JW, Wood MG, Osterman J, Cali JJ,
Daily WJ, Meisenheimer PL, Klaubert DH, (2008) "N-Alkylated 6-Aminoluciferins are bioluminescent Substrates for Ultra-Glo and
QuantiLum Luciferase: New Potential Scaffolds for Bioluminescent Assays"Biochemistry 47:
10383–10393.
- Branchini BR, Murtiashaw MH, Magyar RA, Portier NC,
Ruggiero MC, Stroh JG (2002)"Yellow-Green and Red Firefly Bioluminescence from 5,5-Dimethyloxyluciferin." J. Am. Chem. Soc. 124: 2112–2113.
- White EH, Worther H, Seliger HH, McElroy WD, (1966) "Amino Analogs of Firefly Luciferin and Biological Activity Thereof." J. Amer. Chem.
Soc. 88: 2015-2019.
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