A High Throughput In Vivo Model for Testing Delivery and Antiviral Effects of siRNAs in Vertebrates  Brian Dall Schyth, Niels Lorenzen, Finn Skou Pedersen  Molecular Therapy  Volume 15, Issue 7, Pages 1366-1372 (July 2007) DOI: 10.1038/sj.mt.6300150 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Verification of small interfering RNA (siRNA) functionality in human embryonic kidney 293T cells. (a) Two different siRNAs, siVHSV-G193 and siVHSV-G537, targeting the viral hemorrhagic septicamia virus glycoprotein (VHSV-G) were transfected with the expression vector pcDNA3-vhsG, which encodes VHSV-G. (b) Suppression of VHSV-G as evaluated by real-time polymerase chain reaction was approximately 75–80% compared with mock control cells treated with the transfection agent only. An siRNA targeting enhanced green fluorescent protein (EGFP) (Dharmacon) was used as specificity control. G protein intensity in 293T cells was evaluated by immunofluorescence of cells stained using rhodamin and antibodies specific to VHSV-G. Scale bar = 60 μm. (c) The specificity of siRNAs was also tested by transfection with an expression vector encoding the glycoprotein of the heterologous rhabdovirus infectious hematopoietic necrosis virus (IHNV). In all cases values are mean G messenger RNA levels from three wells normalized to the mock control + SD. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 In vivo uptake of fluorescein isothiocyanate–conjugated small interfering RNAs (siRNAs). Twenty-four hours after intraperitoneal (IP) injection of rainbow trout with 10 μg siRNA per gram body weight, 10-μm cryosections were made, counterstained with 4′-6-diamindino-2-phenylindole (DAPI), and examined. (a–d) The same area of the tissue section using a fluorescein isothiocyanate filter to detect fluorescent siRNAs (upper row) and a filter for DAPI (lower row) to visualize and identify specific tissues in the sections. Intestinal lumen (il), muscle tissue (m), liver (l), kidney (k), and IP cavity (ic). (a) Anterior of ic dorsal to intestine with fluorescent siRNAs mainly residing in cells lining the ic ventral to the kidney. (b–c) Fluorescent siRNAs were also seen in cavities between intestinal lobes. (c) Close-up of the space between intestinal blind sacks showing location of fluorescent cells. (d) Ic sections from fish injected with naked fluorescent siRNAs and (e) non-fluorescent 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–formulated siRNAs, respectively, were used as controls. (f) Fluorescent siRNAs in the cytoplasm of an IP cell captured at ×100 magnification. Four fish per group were included in the analysis. Representative findings are displayed. Scale bars (a, b, d, e) = 1 mm, (c) = 500 μm, (f) = 40 μm. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Free intraperitoneal (IP) cells harvested 24 hours after IP injection of (a) naked fluorescent small interfering RNAs (siRNAs) or (b) 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–formulated fluorescent siRNAs, respectively, as described in Materials and Methods. Micrographs in the left column were taken by light microscopy and pictures to the right using a filter for detection of fluorescein isothiocyanate. Cells were harvested and inspected from three fish per group. Scale bars = 15 μm. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Effect of intraperitoneal (IP) small interfering RNAs on development of mortality among rainbow trout fry following challenge with viral hemorrhagic septicemia virus (VHSV). (a) At day-1 fish were IP-injected with 0.9% NaCl, naked small interfering RNAs, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–formulated siVHSV-G193 and siVHSV-G537, respectively. At day 0, virus was added to a final concentration of 105 tissue culture infectious dose50/ml in the aquarium water followed by daily monitoring of mortality. (b) An extended experiment included fish injected with DOTAP, fish injected with the DOTAP-formulated siRNA against enhanced green fluorescent protein (EGFP), and fish injected with two different doses of the DOTAP-formulated siVHSV-G193. In both experiments 30 fish were used per condition. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Expression of the interferon (IFN)-induced antiviral guanosine triphosphatase Mx3 in rainbow trout liver following intraperitoneal (IP) injection of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/small interfering RNAs (siRNA) formulations. RNA was purified from the liver of fish 48 hours after injection with the DOTAP/siRNA formulations. (a) Cryosections of liver counterstained with 4′-6-diamindino-2-phenylindole (top) show that delivery of siRNAs to this organ was below the detection limit. Scale bar = 1 mm. (b) Mx3 expression analysis by real-time polymerase chain reaction on liver tissue from fish IP-injected with polyinosinic:polycytidylic acid (polyI:C) (positive control), DOTAP control, the three DOTAP/siRNA formulations used throughout this study, and naked siVHSV-G193. Owing to the large variation in the expression data, groups were compared according to their median rather than their mean. N denotes the number of fish in each group. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 The delivery agent 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) is able to increase the interferon-stimulatory potential of the toll-like receptor 3 ligand polyinosinic:polycytidylic acid (polyI:C). This concept was tested in rainbow trout (a) in vivo and (b) in vitro using rainbow trout gonad cells (RTG2). Trout were injected and processed as in the experiment corresponding to Figure 5. Injected reagents were polyI:C and DOTAP-formulated polyI:C, using saline and DOTAP alone as controls. Cell layers were transfected by the same reagents using only the DOTAP control. Expression analysis is as in Figures 1 and 5. Mean value from (a) four fish + SD or (b) three cell culture wells + SD. Readout for DOTAP alone was in both cases used for normalization. Molecular Therapy 2007 15, 1366-1372DOI: (10.1038/sj.mt.6300150) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions