YFP expression following transduction of primary cultures of chick embryonic cells with FIV-YFP vectors.

<p>Primary cell cultures from E11 chick embryos were transduced with FIV-YFP (2×10⁴ TU/well, in a 24-well plate) and YFP-expressing cells (green) were detected by fluorescence microscopy, 48 h after transduction (<b>A–F</b>). The corresponding bright-field micrographs of the same cultures (<b>A</b>′<b>–F</b>′) demonstrate the tissue-specific morphology of the cells. Scale bar = 20 µm. Rate of transduction in each culture, determined by FACS analysis, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036531#pone.0036531.s002" target="_blank">Figure S2</a>.</p

Estimation of transduction efficiency in livers of <i>in ovo</i> FIV-YFP transduced chicks by flow cytometry.

<p>Liver samples from 2-day-old control chicks treated with PBS (<b>A</b>) or FIV-YFP (<b>B</b>) were analyzed by flow cytometry. One representative image from the control PBS and FIV-YFP treated chick groups is shown. Cells in each sample were selected (window P1 in A) and YFP fluorescence was analyzed (B). While no obvious signals were obtained in the control samples, the rate of transduced liver cells in the samples from FIV-YFP treated chicks was 0.46±0.19% (n = 3). Blue and red mark the YFP-positive and negative cells, respectively. The sample with the highest transduction rate (0.8%) is presented.</p

Serial paraffin sections of YFP-positive cell cluster in the liver.

<p>A cluster of YFP-expressing cells (green) was identified in five consecutive liver sections (<b>A–E</b>) by immunofluorescence with anti-GFP antibody, demonstrating its 3D structure. DAPI staining (blue) indicates cells nuclei. Arrowheads in <b>E</b> indicate nuclei of transduced cells. Scale bar = 20 µm.</p

FIV-α-MSH transduction of HEK293T cells: characterization at the DNA, mRNA and protein levels. A

<p>. Genomic DNA extracted either before (293T) or 24 and 72 h after transduction of HEK293T cells with FIV-α-MSH (293T-α-MSH) was analyzed by PCR using the recombinant α-MSH-specific primers. The plasmid pLionII- α -MSH was used to demonstrate the expected size fragment. <b>B</b>. RNA samples, prepared from similarly treated cell cultures 20 days after transduction, were analyzed by RT-PCR using the same primers. <b>C</b>. The integrity of the mRNA in the control and transduced culture cells was demonstrated using the GAPDH primers. <b>D</b>. Production and secretion of α-MSH peptide was demonstrated by RIA. α-MSH peptide was detected in cell lysate (L) and culture medium (CM) of HEK293T cells, 10 days after transduction with FIV-α-MSH. No signal was detected in cell lysate or culture medium of control HEK293T cells, transduced with FIV-YFP (293T-YFP).</p

Viability of transduced cells in liver of chicks, as estimated using TUNEL and PCNA.

<p>Liver paraffin sections of FIV-treated chicks (<b>A</b>) and controls (<b>B</b>), immunostained with anti-GFP antibody (green) and DAPI (blue), were analyzed for apoptosis using TUNEL assay (A′, B′, red). Very low incidence of cell death was observed in both groups of chicks. The areas of the TUNEL positive cells were enlarged in the white boxes (inserts). Scale = 100 µm. Liver paraffin sections were immunostained with anti-GFP and DAPI (C, green and blue, respectively) and anti-PCNA (C′, red). Co-labeling of a group of hepatocytes is demonstrated (C′′), indicating actively proliferating FIV-transduced cells. Scale = 50 µm.</p

α-MSH expression in tissues of FIV-α-MSH-treated chicks.

<p>qPCR analysis of the indicated tissues from 2-day post-hatch chicks was performed following <i>in-ovo</i> treatment with FIV-α-MSH particles (2×10⁶ TU/chick), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036531#pone-0036531-g002" target="_blank">Figure 2</a>. Results of transgene expression in tissues of each chick are indicated by a different color (n = 6). Spleen and liver tissues of chicks treated with FIV-YFP particles served as negative controls. Expression of α-MSH in the liver is also demonstrated in 40-day-old chickens (n = 4). A similar analysis performed using tissues of 2-day-old chicks treated with FIV-YFP, but with YFP-specific primers, is shown in the insert (n = 4). A higher level of transgene expression in the liver and spleen was common to both transgenes. The results are presented as mean ± SEM of triplicate runs (error bars are contained within the symbols).</p

CR1-PCR analysis for genomic integration of YFP in chicken cells, transduced with FIV-YFP vectors.

<p>Schematic representation of the two-step PCR approach using the CR1- and LTR-specific primers. Diluted (1∶1000) mixes from the first long PCRs, employing CR1 and LTR primers, were used as templates for short nested PCRs with nested LTR primers. In the absence of genomic integration, no signals are expected in the nested PCR. This is indicated by the control long PCR with no CR1 primers. <b>B</b>. Nested PCR products separated on 2% agarose gel, obtained using as templates the first long-PCR DNAs, which were prepared 14 days after <i>in vitro</i> transduction with FIV-YFP, from E11 liver and muscle cells. The primers used for the first long PCR are indicated: the LTR primers were 5′LTR (5′L) and 3′LTR (3′L); the CR1 primers were CR1-1F (C1F), CR1-1R (C1R), CR1-3F (C3F) and CR1-3R (C3R). The nested primers are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036531#s2" target="_blank">Materials and Methods</a>. Neither the controls without CR1 primers, nor the control using DNA from non-transduced cell cultures gave a signal. The expected size fragments were 120 bp for the 5′LTR and 110 bp for the 3′LTR. The products were confirmed by sequencing. These results indicate genomic integration of FIV-YFP-derived cDNA in the host chicken cells. M, molecular weight markers.</p

Immunofluorescence analyses of YFP expression in the liver and spleen of post-hatch chicks following <i>in ovo</i> FIV administration.

<p>Whole-mount immunostaining with anti-GFP antibody (green) and anti-SMA antibody (red) was performed on pieces of livers from: 2-day-old chicks treated with either PBS (<b>A</b>) or FIV-YFP (<b>B</b>), and from 40-day-old FIV-YFP-treated chicks (<b>C</b>). Images were obtained using epi-fluorescent stereomicroscope. Scale bar = 200 µm for A and C, and 0.5 mm for B. Immunostaining of paraffin sections with anti-GFP antibody was performed on liver (<b>D & E</b>) and spleen (<b>F & G</b>) tissues from 2-day-old chicks treated with FIV-YFP. For each of these sections, DAPI staining (blue) is shown in the corresponding images (<b>D′–G′</b>) to indicate cell nuclei. Arrowheads mark nuclei of YFP-expressing cells with splenocyte morphology (F′). Merged YFP and DAPI staining is shown in the corresponding <b>D′′–G′′</b>. Images were obtained by confocal microscopy except for <b>E</b>, <b>E′</b> and <b>E</b>′′, which were obtained by epi-fluorescenct microscope. The arrow in <b>E′′</b> indicates a YFP-expressing cell with endothelial morphology located next to transduced cells with hepatocyte morphology. <b>H</b>. Confocal images of whole-mount immunostained liver sample, using both anti-GFP (<b>H</b>) and anti-SMA (<b>H′</b>) antibodies, confirmed the association of some of the YFP-expressing cells with blood vessels, which appear in yellow in the merged image (<b>H′′</b>). Scale bar = 20 µm (D-G) and 50 µm (H).</p