Volume 9, Issue 4, Pages 617-624 (April 2004) Use of adenovirus protein IX (pIX) to display large polypeptides on the virion— generation of fluorescent virus through the incorporation of pIX-GFP  Robert A Meulenbroek, Kathy L Sargent, John Lunde, Bernard J Jasmin, Robin J Parks  Molecular Therapy  Volume 9, Issue 4, Pages 617-624 (April 2004) DOI: 10.1016/j.ymthe.2004.01.012 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Schematic representation of Ad vectors used in this study. Ad/pIX is deleted of the Ad early region 1 (E1) and E3 regions and encodes a C-terminal FLAG-tagged pIX under endogenous regulation. Ad/pIX-GFP is similar to Ad/pIX, but encodes a pIX-GFP fusion, as described under Materials and Methods. AdCMV-GFP is deleted of E1 and E3 and encodes GFP under the regulation of the human cytomegalovirus immediate-early promoter/enhancer and bovine growth hormone polyadenylation sequence (not shown). pIX in this vector is unaltered. AdRP2309a is a helper virus for use in the Cre/lox system for generating hdAd and encodes a pIX-GFP fusion protein as described for Ad/pIX-GFP. AdRP2309a contains a floxed packaging signal, is deleted of E1, contains the E4 open reading frame 6 (E4orf6) under the regulation of the mouse mammary tumor virus long terminal repeat within the E3 region, and is deleted of the native E4orf6, as has been previously described [4]. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Expression of pIX in vitro. A549 cells were transiently transfected with plasmid expressing pIX-GFP (A–C), GFP (D–F), or pIX-FLAG (G–I). Twenty-four hours posttransfection, the cells were fixed and stained with Hoechst to visualize the cell nuclei. For cells transfected with pIX-FLAG, the protein was detected using immunofluorescence with an anti-FLAG antibody. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Fluorescent visualization of Ad/pIX-GFP. Virus was propagated in 293 cells, and the virus was purified by cesium chloride buoyant density centrifugation, as described under Materials and Methods. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Incorporation of pIX-GFP in the Ad virion. Ad/pIX or Ad/pIX-GFP (2.5 × 107 pfu) was heated in SDS–PAGE protein loading buffer and separated by 15% PAGE. The resulting proteins were transferred to a PVDF membrane and probed with antibody to FLAG (top) or Ad 5 fiber (bottom). The bands corresponding to pIX and pIX-GFP are indicated with arrows. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 In vitro visualization of Ad/pIX-GFP. A549 cells were infected with AdCMV-GFP or Ad/pIX-GFP at a multiplicity of infection of approximately 800 particles per cell and, 10 or 40 min later, the cells were fixed and stained with Hoechst, and GFP fluorescence was visualized by microscopy. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 In vivo visualization of Ad/pIX-GFP. Top: Adult C57Bl/6J mice were injected in the tibialis anterior muscle with 2.5 × 107 pfu of Ad/pIX-GFP. Thirty minutes later, the muscle was removed, embedded in OTC, and frozen in melting isopentane precooled by liquid nitrogen. Sections of the tissue were prepared and fluorescence was visualized by microscopy. Bottom: To examine the pathology of injected muscle, tissue sections were stained with hematoxylin and eosin. Molecular Therapy 2004 9, 617-624DOI: (10.1016/j.ymthe.2004.01.012) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions