sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase敏感的双重体内生物发光成像使用新的红色密码子优化的萤火虫荧光素酶和绿色磕头虫荧光素酶.pdfVIP

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase 1 2 2 3 1 ¨ 2 Laura Mezzanotte , Ivo Que , Eric Kaijzel , Bruce Branchini , Aldo Roda *, Clemens Lowik * 1 Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy, 2 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands, 3 Department of Chemistry, Connecticut College, New London, Connecticut, United States of America Abstract Background: Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color- coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings: Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spec