Marker Gene Monthly Newsletter - Volume 11, Number 4 - April, 2011

Luciferase as a Tracking Tool for Tumor Cells and the Associated Immune Response.

Subcutaneous models of cancer and tumor development have become essential in the search for new drug candidates with therapeutic potential and to visualize the location and growth of tumors in vivo upon treatment. The use of the firefly luciferase gene (luc) as a marker for cells in animal xenograft models has been well documented as the luminescence can be detected deep into living tissue using sensitive photographic CCD camera techniques. In addition, by injecting tumor cells into various tissues, the potential efficacy of a treatment regimen on various locations can be evaluated and used to predict the outcome of drugs in later clinical trials. Such mouse models have recently been used by researchers at the British Columbia Cancer Agency to monitor growth of human breast cancer cells transfected with a luciferase gene and to study the efficacy of treatment with docetaxel in vivo. In these experiments female mice are injected with the human breast cancer cells at various locations to create different manifestations of disease, the infected mice are then treated with the drug that is being studied and the growth of the tumor is monitored by bioluminescence imaging.
Similarly work has been done at the M.D. Anderson Cancer Center in order to track the immune response to cancer cells by transfecting T cells with an enhanced version of the firefly luciferase gene that generated up to 100 times more light than genes used in previous studies. This allowed monitoring or distribution and trafficking of as little as 10 T cells to be visualized within the mouse after transplantation. The tracking of T cells could also then allow the location of tumors in an adoptive immunotherapy model to be elucidated. To find out more about these techniques, please see the references below or visit our website.

• Zhang W, Feng JQ, Harris SE, Contag PR, Stevenson DK, Contag CH (2001) "Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression." Transgenic Research 10(5): 423-434.
  
• Kalra J, Anantha M, Warburton C, Waterhouse D, Yan H, Yang Y-J, Strut D, Osooly M, Masin D and Bally MB (2011) "Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267." 11(9): 826.
  
• Rabinovich BA, Ye Y, Etto T, Chen JQ, Levitsky HI, Overwijk WW, Cooper LJ, Gelovani J, Hwu P (2008) “Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.”  Proc. Natl. Acad. Sci. U. S. A. 105(38):14342-14346.
  
  

Use of Green Fluorescent Protein to Study Promoter Activity

Saccharomyces cerevisiae is an important FDA-approved cloning vehicle for production of recombinant proteins, since it is endotoxin free and non-pathogenic. When evaluating promoter activity for gene expression in S. cervisiae, the ability to monitor promoter strength and activity upon induction or repression is important for control and biological efficacy studies. A common way to monitor the promoter activity is to place a reporter gene under control of the regulatory element. Green fluorescent protein (GFP) is particularly useful for this application as it is autofluorescent so there is no need for a substrate, the protein is relatively small and so does not typically interfere with gene function and the protein is also non-toxic to the host cell. The promoter can then be analyzed quantitatively by the amount of green fluorescence measured and the effects of induction and repression studied.
This technique has been employed by researchers at the University of Maryland to track real-time activity of the GAL1 promoter in yeast cells. This promoter is sensitive to the concentration of certain sugars in the cell, it is repressed in the presence of glucose and induced when glucose is absent and galactose present in the cell. By cloning a GFP gene downstream of the GAL1 promoter the group was able to show that an increase in galactose concentration led to a proportional increase in green fluorescence measurable in the cells. The use of GFP as a reporter of promoter activity has been taken further by the same group for use with E. coli  in a 96-well format, where different amount of inducer or repressor was administered in a high throughput format. In this work the strong promoter pBAD was compared to weaker promoters sodA and acnA. pBAD is induced by increasing levels of glucose and arabinose in the cell, whereas the weak promoters could be induced by the drug paraquat, which induces oxidative stress. These experiments showed that all 3 promoters could be induced in a dose-dependent 96-well format and the level of induction monitored using GFP fluorescence. The application of these methods to general promoter strength or induction assays in either S. cervisiase or E. coli are significant. For more information about these methods, please see the references below, or visit our website.

• Li J, Wang S, VanDusen W J, Schultz LD, George HA, Herber WK, Chae HJ, Bentley WE Rao G (2000) “Green fluorescent protein in Saccharomyces cerevisiae: Real-time studies of the GAL1 promoter.”  Biotechnology and Bioengineering 70: 187–196.
  
• Lu C, Bentley WE, Rao G  (2004) “A High-Throughput Approach to Promoter Study Using Green Fluorescent Protein.”  Biotechnology Progress  20: 1634–1640.
• Tsien RY (1998) ”The green fluorescent protein.” Ann. Rev. Biochem. 67: 509-544.

OliGlo^TM Fluorescent In Situ Hybridization Kits-Fishing for Success.

Fluorescent In Situ Hybridization (FISH) is a powerful technique in many areas of life science both as a research and a diagnostic tool. By hybridizing labeled probes that are complimentary to the gene of interest the region of the chromosome where that gene is located can be visualized. For example FISH can be used in the diagnosis of Down Syndrome. Probes are designed to hybridize to chromosome 21 and the number of chromosomes can then be counted. This has an advantage over traditional karyotyping for diagnosis in that the presence of a third chromosome can be seen instantly without the need for time consuming pairing of chromosomes.
The OliGlo™ FISH kit has been specially designed to provide the researcher with everything they will need to label probes for hybridization. Due to the specific properties of the dye binding to the probe the labeling occurs on the phosphate backbone of the probe preventing disruption of hybridization due to labeled bases being unable to hybridize to their complement. This allows for non sequence dependent highly sensitive labeling. The kits are available now in 2 sizes and 3 different detection agents, with free domestic shipping until the 31st of May. To find out more, please see the references below or visit our website.

• Forster AC, McInnes JL, Skingle DC, Symons RH. (1985) "Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin." Nucleic Acids Res 13: 745-791.
• Liu P, Siciliano J, Seong D, Craig J, Zhao Y, Jong P, Siciliano M. (1993) "Dual Alu polymerase chain reaction primers and conditions for isolation of human chromosome painting probes from hybrid cells." Cancer Genetics Cytogenetics 65:93-99.
• Henegariu O, Bray-Ward P, Artan S, Vance G, Qumsyieh M, Ward D. (2001) "Small marker chromosome identification in metaphase and interphase using centromeric multiplex FISH (CM-FISH)." Lab Investig 81(4): 475-481.
• Weier H, Lucas J, Poggensee M, Segraves R, Pinkel D, Gray J. (1991) "Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations." Chromosoma 100:371-376
• Lichter P, Cremer T, Tang CJ, Watkins PC, Manuelidis L, Ward DC (1988) “Rapid detection of human chromosome 21 aberrations by in situ hybridization” PNAS 85(24): 9664-9668

Introducing our New Lab-Safe Packaging!

It's springtime and time to bring in the new. At Marker Gene we have done just that with a set of new custom-designed boxes and labels to package our products and kits. These new labels have been tested to be fully waterproof for use in freezer storage, are resistant to a variety of lab solvents, and even after multiple freeze-thaw cycles remain looking crisp and neat. The new boxes are made from sturdy laminated materials making them durable for laboratory storage and more than just a means of transport. They can also be reused over and over for routine applications. Order your kits and reagents today and see our new packaging for yourself!

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