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Development is an orderly process of events by which an organism or part of an organism acquires its final form and function. At the molecular level, this is reflected in pathways of genetic events that also must occur in an orderly fashion in order for development to proceed normally. Our lab uses conditional gene expression in the transgenic mouse to explore the consequences of disrupting these pathways during development and the mechanisms by which such disruptions can cause disease. Within this general framework, members of our lab work on a limited-but diverse-group of human disorders using the tools of experimental genetics and cell and molecular biology. We are studying how mutations in the tumor suppressor p53 can limit both growth and lifespan by impinging on one of the major molecular pathways that controls energy metabolism. And, we are exploring how a p53-like pathway might contribute to AIDS pathogenesis. Another major focus of the research in the lab is on the mutant protein that causes Huntington's disease, a neurological disorder resulting from the death of neurons in specific regions of the brain. You can read more about these projects by following the links to the individual members of the lab. Central to our work is the construction and utilization of a system of conditional gene expression that allows us to turn specific genes on and off in the mouse (Genes & Development 15:1506-1517). The mouse is the most widely used experimental animal to model mammalian development and disease. In recent years, technological advances in genetic manipulation of the mouse genome have made it even more valuable as a research tool, and a great deal of information has been learned from conventional knock-out and transgenic experiments. Tight, reversible control of gene expression greatly broadens the possible experimental questions that can be addressed with mouse models. If you would like to hear an interview with CBC's "Quirks and Quarks" discussing the kinds of questions that can be addressed, click here. If you would like to see a video discussing our techniques, click here. (The video was produced by Science and Technology News Network). Our system is based on the lac operon of E. coli, a segment of the bacterial genome that encodes the enzymes necessary for lactose metabolism. To conserve energy, the genes encoding these enzymes are transcribed only when lactose is available as a food source, and are silenced in its absence. Lactose interacts with a regulatory protein (the lac repressor) and its DNA binding sequence (the lac operator) to switch the expression of genes in the lac operon on and off. We use the lac repressor and lac operator to control the expression of genes in the mouse in an analogous way.
Unlike the other currently available gene regulatory systems for the mouse, the lac system allows for direct, reversible control of mammalian promoters, so that the expression levels achieved from the target gene are physiological. An additional benefit of the system is that directly targeting the lac repressor to operator sequences incorporated into mammalian promoters completely eliminates the necessity of using viral promoters or viral DNA binding proteins in conjunction with a prokaryotic-based regulatory system. A target gene made up entirely of mammalian elements is more likely both to maintain functionality after transiting embryogenesis and to be expressed in the appropriate tissue at the appropriate level. This lends the system a particularly strong element of predictability that other prokaryotic-based systems cannot match. Individual members of the lab are using the lac operator-repressor system in the mouse to explore different molecular pathways important during development and disease. Amy Ryan is generating a novel mouse model of Huntington's disease in which a full-length mutant HD gene will be under the control of the lac repressor. The model will allow us to ask questions about the progressive nature of HD by defining a window of vulnerability when the mutation causes the most severe consequences. Dietlinde Wolf is applying the lac operator/repressor system to the study of HIV pathogenesis. The system will allow her to turn on viral gene expression at an immunologically appropriate time in the animal's life. Tsutomu Sasaki is developing a system of conditional RNAi expression for the mouse that can be used to knock down the level of specific proteins. One possible application might be to target a mutant gene product when both the mutant and normal proteins are found in the same cell, as is the case with HD. Another target might be a mutant form of the tumor suppressor, p53. Wendy Gluba has derived a line of mice with a N-terminal deletion of p53 that limits both size and life-span. The phenotype is only seen when both mutant and wild-type forms of p53 are present in the same cell. Bernhard Maier is exploring the hypothesis that p53 exerts control on cellular metabolism and proliferation by regulating the activity of the IGF pathway. He is assisted by our "Master of the Genome," Brian Bernier . Brian is also constructing a regulatable reporter transgene using green fluorescent protein that will be used to characterize lac repressors with cell- or developmental stage-specific expression patterns that are being generated by Susan Prasad . Finally, the consequences of disrupting p53 or HD in the brain are being characterized by Silvia Medrano . To visualize their effects, she has developed a mouse in which changes in synaptic activity trigger luciferase activity; the light emitted in the process can be detected and imaged in the living animal. To learn more about Heidi Scrable, click here . Some former members of the lab include Dr. Carolyn Cronin , who has just finished her clinical training for the combined MD/PhD degree, Dr. Ian McCaffery , currently a project leader at Celera Genomics, Dr. Lee Ligon, currently a postdoctoral fellow at the University of Pennsylvania, and Kathryn Edwards and Liana Abramova, currently medical students in Ireland and at Emory University, respectively. We still miss them. |
