Using osteoclast differentiation as a model for gene discovery in an undergraduate cell biology laboratory.

ABSTRACT: A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown" expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.

SUBMITTER: Birnbaum MJ 

PROVIDER: S-EPMC4090094 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature