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a1-Proteinase Inhibitor Promoter Directed LacZ Expression in the Mouse Cornea.

The ability to confer gene expression to individual tissues or organs in vivo presents itself as an exciting approach to treating congenital defects and genetic diseases by gene therapy. But directing the expression of exogenous genes within the organism continues to be an area of active research. Recent work from the laboratory of Professor Beatrice Yue in the Department of Ophthalmology and Visual Sciences at the University of Illinois-Chicago College of Medicine has described a new method of targeting gene expression to sections of the mouse cornea by using the 1.4-kB alpha 1-proteinase inhibitor promoter. Expression was monitored using the lacZ b-galactosidase marker gene, and analysis by immunohistochemistry. The a1-Proteinase inhibitor (a1-PI) is a major protease inhibitor found in human serum. One of its primary physiological roles is to protect the elastic fibers in lung alveoli from excessive digestion by neutrophil elastase activity. Defective expression of this protein in lung tissue is a primary cause of degenerative lung disease. But it is also found expressed in the liver and in the cornea of the eye. Under-expression in the eye has been linked to keratoconus, a cornea degradative disease. Using specific transcription factor analysis, a minimal promoter sequence was developed that was found to target lacZ expression preferentially in the epithelium and stroma of the mouse cornea, with minimal activity in white blood cells, skin or brain. The a1-PI-lacZ construct was microinjected into C57BL/6 X SJL F2 hybrid mouse eggs. Transgenic mice appeared to be normal with eyes normal in size and morphology for up to three progenies. Examination of the b-galactosidase reporter gene activity by histochemical (X-Gal) staining resulted in only very faint staining in the cornea. But this relatively weak activity may be an advantage in that a foreign gene, when introduced using this method into corneal tissues, should not induce side effects from over-expression. This type of tissue specific expression in both the corneal epithelium and stroma may be very useful for future gene manipulation studies, for the evaluation of epithelial-stromal interactions in wound healing, and for studies of corneal diseases such as keratoconus, a cornea-thinning disease. For more information about these and similar tissue specific targeting approaches, please see our website or the references below.

• Ueda J, Li Y, Goh MS, Maruyama Y, Sugar J, Yue BYJT (2002) "Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice." Anat. Rec. 266(1):5-9.
• Li Y, Zhou L, Twining SS, Sugar J, Yue BYJT (1998) "Involvement of Sp1 element in promoter activities of the a1-proteinase inhibitor gene. J. Biol. Chem. 273:9959–9965.
• Liu CY, Arar H, Kao C, Kao WWY. (2000) "Identification of a 3.2 kb 59-flanking region of the murine keratocan gene that directs b-galactosidase expression in the adult corneal stroma of transgenic
  mice. Gene 250:85–96..
• Kays WT, Piatigorsky J. (1997) "Aldehyde dehydrogenase class 3 expression: identification of a cornea-preferred gene promoter in transgenic mice." Proc Natl Acad Sci USA 94:13594–13599.
  

Triple Marker Gene System for Whole Body Analysis.

Marker gene imaging has found a wide application in the development and monitoring of different adoptive cell and gene therapies. These systems provide both spatial and temporal analysis dimensions to the understanding of different molecular-biological processes and has been termed “molecular-genetic imaging”. Researchers at the Memorial Sloan Kettering Cancer Center in New York recently developed a triple marker gene system (TGL) for monitoring gene transfection with whole body analysis using a combined thymidine kinase (tk), green fluorescent protein (GFP) and luciferase (luc) retroviral expression system. This triple modality system provides fluorescent, chemiluminescent and enzymatic analysis of expression levels in vivo using whole-body PET, fluorescence and biolumininescence imaging of TGL transduced xenografts. The thymidine kinase activity was monitored by PET using radio-iodinated [¹³¹I] 1-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)-5-iodouracil (FIAU). Luciferase activity was measured using whole body bioluminescence using a Luminoscan Ascent camera system after administration of D-Luciferin. And whole body fluorescence could be monitored using a hand made digital optical fluorescence imaging (OFI) system consisting of a 150-W halogen light source LT-9500 equipped with an excitation filter (480–490 nm), two fiberoptic guides with linear illuminators (each positioned at 45°), and an emission filter plate (505 nm cut-off filter; 6x12 cm). The GFP fluorescence could be readily seen with the naked eye, and imaged with a CCD camera. The individual marker genes were also measured in vitro in cell culture prior to implantation.

Cross-comparison and validation of the results obtained by these various in vitro, in situ, and in vivo imaging modalities indicated excellent correlation for tranplanted transgenic tumor tissues in a mouse model, with the tk-induced PET, SPET, gamma camera analyses showing better spacial resolution than for the GFP fluorescence or luciferase bioluminescence methods. For more information about these multiplexed marker gene approaches, please see our website or the references below.

• Ponomarev V , Doubrovin M , Serganova I, Vider J, Shavrin A , Beresten T, Ivanova A, Ageyeva L, Tourkova V, Balatoni J, Bornmann W, Blasberg R, Tjuvajev JG (2004) "A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging." Eur. J. Nucl. Med. Mol. Imaging 31:740–751.
• Yang M, Baranov E, Moossa AR, Penman S, M. Hoffman RM (2000) "Visualizing gene expression by whole-body fluorescence imaging." Proc. Natl. Acad. Sci. USA 97(22): 12278-12282.
• Green L, Yap C, Nguyen N, Barrio J, Namavari M, Satyamurthy N, Phelps M, Sandgren E, Herschman H, Gambhir S (2002) "Indirect monitoring of endogenous gene expression by positron emission tomography (PET) imaging of reporter gene expression in transgenic mice." Mol. Imaging Biol. 4:71–81.
• Cowsill C, Southgate TD, Morrissey G, Dewey RA, Morelli AE, Maleniak TC, Forrest Z, Klatzmann D, Wilkinson GW, Lowenstein PR, Castro MG (2000) "Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK." Gene Ther. 7:679–685.

LacZ Cre-Recombinase Systems.

For analysis of positive lacZ expression in these systems, simple X-Gal staining can be used, but much more sensitive analysis can be accomplished by utilizing the FACS-Gal analysis technique, which involves either tissue staining or FACS-based detection of lacZ+ cells with the fluorogenic substrate, FDG, of galactosidase (the lacZ enzyme). b-Galactosidase cleaves FDG and releases a fluorescent product fluorescein, which is several orders of magnitude more sensitive than X-Gal analytical methods. However, it is common that the lacZ signal can vary from sample to sample. Recently a procedure to ensure reliable and consistent results for the whole hematopoietic system, with the exception of terminally differentiated erythroid cells, or red blood cells that express much lower levels of b-Gal, was developed in the laboratory of Professor Hong Wu at UCLA. For more information about these methods of analysis, please visit our website or see the references below.

• Klaus Rajewsky, Hua Gu, Ralf Kühn, Ulrich A.K. Betz, Werner Müller, Jürgen Roes, and Frieder Schwenk (1996) "Conditional Gene Targeting." J. Clin. Invest. 98(3): 600–603.
• Soriano P, (1999) "Generalized lacZ expression with the ROSA26 Cre reporter strain." Nat. Genet. 21:70-1.
• Mao X, Fujiwara Y, Orkin SH (1999) "Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice." Proc. Natl. Acad. Sci. U S A 96:5037-42.
• Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S (1996) "Subregion- and cell type-restricted gene knockout in mouse brain." Cell 87:1317-26.
• Sato M, Yasuoka Y, Kodama H, Watanabe T, Miyazaki JI, Kimura M (2000) "New approach to cell lineage analysis in mammals using the Cre-loxP system. Mol. Reprod. Dev. 56:34-44.
• Guo W, Wu H, (2008) "Detection of LacZ expression by FACS-Gal analysis." Nature Protocols DOI: 10.1038/nprot.2008.163.

New Anerobic GFP Proteins for Expression in Bacteria and Yeast.

• Drepper T, Eggert T, Circolone F, Heck A, Krau U, Guterl JK, Wendorff M, Losi A, Gartner W, Jaeger KE (2007) "Reporter proteins for in vivo
  fluorescence without oxygen." Nat. Biotechnol. 25: 443-445.
• Losi A, Polverini, E, Quest, B, Gartner, W. (2002) "First Evidence for Phototropin-Related Blue-Light Receptors in Prokaryotes." Biophys. J. 82: 2627–2634.
  
• Krauss U, Losi A, Gartner W, Jaeger KE, Eggert T (2005) "Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct."Phys. Chem. Chem. Phys. 7: 2804–2811.
• Buttani, V., Losi, A., Eggert, T., Krauss, U., Jaeger, K.-E., Cao, Z. and Gärtner, W. (2007) "Conformational analysis of the blue-light sensing protein YtvA reveals a competitive interface for LOV-LOV dimerization and interdomain interactions." Photochem. Photobiol. Sci. 6: 41-49.

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• 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.
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