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

Abstract

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.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares

“…For all recombinant viruses, recombinant plasmids were first constructed using the MultiSite Gateway Pro system (Invitrogen), as described previously ( 27 , 39 , 40 ), or the Gibson cloning method (New England BioLabs, USA). Upstream and downstream hybridizing sequences were amplified by PCR using specific primers to create entry clones by the Gateway BP recombination system with appropriate pDONR vectors.…”

Self-assembling functional programmable protein array for studying protein–protein interactions in malaria parasites

“…The product obtained from each gene was purified by Wizard SV Gel and PCR Clean-Up System (Promega), according to the manufacturer’s specifications. The second round of PCR involved 50 μL reaction including a set of universal primers (5 μM concentration), 25 μL 2× KAPA HiFi Ready Mix, 15 μL nuclease-free water and the purified product obtained in the first PCR to introduce the remainder of the attB sequence needed for recombination using the Gateway system (Invitrogen) [ 40 ]. The product obtained from each gene was purified and then used for BP recombination.…”

“…The array surface was blocked with 20 mL Superblock PBS for 1 h at room temperature (RT) with gentle shaking followed by a 5 min wash with deionised water. The arrays were dried under a stream of filtered compressed air [ 40 ]. Blocked slides were incubated with 150 μL/slide of 1:600 (v/v) diluted PicoGreen dye (Invitrogen Inc.) for staining the cDNA to evaluate DNA printing quality.…”

Poxviruses as Gene Therapy Vectors: Generating Poxviral Vectors Expressing Therapeutic Transgenes

“…The Gateway cloning system allows for the simple, rapid, and highly efficient insertion of “att” bounded sequences into a destination vector using a recombinase derived from lambda phage. Smallwood et al has provided an excellent and detailed protocol [67].…”