Engineering a recombinant adenovirus system for the in vitro expression of ?-myosin

Undergraduate Honors Thesis

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/jh343s78m

Abstract

Over three hundred mutations in the molecular motor, myosin, cause a variety of cardiac and skeletal myopathies. One of our goals is to understand the functional implications of these mutations on the myosin molecule. Because myosin motors require a muscle cell context to be functional, the expression system for producing these proteins involves infecting differentiated muscle cells with recombinant adenoviruses. However, the growth of these adenoviruses in nonmuscle cells is limited by an apparent toxic effect of myosin-production in HEK 293 cells during viral expansion. Thus adenovirus generation is the rate-limiting step in this process. To ameliorate this low adenovirus yield, we designed a system of repressing transgene expression in the virus packaging cell line by incorporating microRNA targets into the 3' UTR of the myosin transgene. As a proof of principle, using the kidney-specific miRNA, miR-192, multimers of the miR-192 target were introduced into a luciferase reporter through cloning. Subsequent experiments using the miR-192 reporter indicated an unexpectedly low expression of miR-192 in HEK 293 cells. Here, we use miR-192 and an artificial miRNA, miR-1-1sc to optimize the transgene repression in HEK 293 cultures through miRNA expressing cell lines. We expect transgene repression in this new miRNA-expressing, virus-packaging cell line will restore virus yields for generating sufficient β-myosin in vitro.

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