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Production of Myxoma Virus Gateway Entry and Expression Libraries and Validation of Viral Protein Expression

Invitrogen's Gateway technology is a recombination‐based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high‐throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development. Curr. Protoc. Microbiol. 21:14A.2.1‐14A.2.29. © 2011 by John Wiley & Sons, Inc.