Robert J , Jancovich JK .

R15 AI101889 NIAID NIH HHS , R24 AI059830 NIAID NIH HHS

Figure 1. (A) Schematic for generating ranavirus recombinant by site-specific integration of a selection cassette. This cassette consists of a fluorescent reporter gene fused to a drug resistance gene by a short linker that is under the control of a ranaviral or ectopic promoter. This cassette is flanked by a left and right sequence portion (500 bp) of the targeted site and is cloned into a convenient bacterial plasmid. Cells are transfected with the construct using lipofectin and then infected with wt ranavirus to generate homologous recombination. The selection is performed sequentially by virus replication in the presence the drug and then by isolation of fluorescent plaques. (B) Schematic representation of the standardized process to generate a recombination cassette for Ambystoma tigrinum virus (ATV). Primers are designed to amplify the neomycin resistance (GNR) cassette as well as approximately 1000 nt of the upstream (US) and downstream (DS) flanking sequences for each target open reading frame (ORF). Adapter sequence added to the 3′ end of the US sequence and the 5′ end of CMV promoter. In addition, a different adapter is added to the 3′ end of the cytomegalovirus promoter (CMV-GNR) cassette and the 5′ end of the DS sequence. A standardized overlapping PCR protocol assembles the recombination cassette that is then agarose gel purified and re-amplified using primers that truncate the US and DS sequences t. This PCR product is then used to generate a recombinant virus.

Figure 2. Detection of FV3-GFP knock-in mutant expressing GFP reporter under the control of the immediate early 18K promoter during infection in vitro in mammalian cell lines and in vivo in X. laevis tadpoles. (A) BHK cells at 2 h post-infection at permissive (30 °C) temperature; (B) mouse BV2 macrophage-like microglial cells at 24 h post-infection at permissive (30 °C) temperature; (C) mouse sertoli macrophage TM4 at 24 h post-infection at non-permissive (37 °C) temperature; and (D,E) midbrain view of pre-metamorphic tadpole brain at 1 day post-infection at low (D) and higher (E) magnification. (*) Indicates the same melanophore in panel D and E. Images are composite of phase contrast and fluorescence for cells (A–C) and of bright field and fluorescence of the whole-mounted tadpole, taken under a Leica DMIRB inverted fluorescence microscope and Infinity 2 digital camera (objectives ×5/×10/×20; Zeiss). Digital images were analyzed and processed by ImageJ software.

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