A3V-mediated Tet-inducible system robustly transduced a sparse population of NM and NL neurons.

<p>(A) A3V-TRE-EGFP and A3V-RSV-rtTAV16 (total 0.5–1.5 µl, 5×10¹¹ GC/ml each) were injected at E3.5, and Dox was administered at E6.5. (B) Double immunofluorescence labeling for EGFP (green) and NeuN (red). No apparent EGFP signal was detected at E17 in the Dox (−) preparation (upper), while strong EGFP signal was observed both at E9 (middle) and E17 (bottom) in Dox (+) embryos. Scale bars indicate 100 µm. (C and D) Higher magnification views of EGFP-expressing NM neurons at E9 and E17, respectively. Scale bars indicate 20 µm. (E and F) NL neurons at E9 or E17, respectively. Scale bars indicate 20 µm.</p

A3V-mediated gene transfer into the embryonic chick auditory brainstem.

<p>(A) Schematic of a primary ITD detection circuit composed of NM and NL neurons. The ipsilateral NM axons provide a simultaneous input to the dorsal side of the NL neurons, whereas the longer delay lines of contralateral NM axons project to the ventral side of more lateral NL neurons. Because NL neurons function as a coincident detector of binaural synaptic inputs, NL neurons in more lateral positions respond maximally to sounds originating in far contralateral space. (B–E) A3V-RSV-EGFP (0.5–1.5 µl, 1×10¹² GC/ml) was injected into the neural tube at E2.5 (B), E3.0 (C), E3.5 (D), and E4.5(E), and EGFP signal at E17 was analyzed by immunofluorescence labeling of coronal sections. Scale bar indicates 200 µm. (F) A3V transduction rates in the embryonic auditory nuclei were quantified as the percentage of EGFP-expressing cells within total NeuN-positive cells in each nucleus (n = 6). The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s005" target="_blank">Table S5</a>. (G) A3V-RSV-mCherry and A3V-RSV-EGFP (0.5–1.5 µl, 1×10¹² GC each) were injected at E2.5 and at E3.5, respectively. Spatial pattern of A3V transduction at E17 was analyzed by double immunofluorescence labeling for EGFP (green) and mCherry (red). Scale bars indicate 200 µm.</p

Comparison of transduction properties of A3V, AAV2 and LV.

<p>(A) Constructs of A3V-RSV-EGFP, AAV2-RSV-EGFP, and LV-RSV-EGFP. ITR, inverted terminal repeat; RSV, Rous sarcoma virus promotor; EGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; pA, SV40 polyadenylation signal; LTR, long terminal repeat; psi, packaging signal; RRE, Rev-responsive element; cPPT, central polypurine tract. (B) A representative example of EGFP-expressing cultured chicken neural cells after A3V treatment. Scale bar indicates 20 µm. (C–E) A3V-treated chicken neural cells, zebra finch neural cells, and 293T cells, respectively. Upper and lower panels represent the DAPI nuclear staining and EGFP fluorescent images of the same fields of view, respectively. All fluorescent images were taken with the same exposure time. (F–K) As a comparison, fluorescent images of corresponding cultured cells after AAV2 or LV treatment are shown. All images were taken with the same exposure condition as in (C–E). Scale bar indicates 100 µm. (L and M) Quantification of overall gene expression and transduction rate, respectively (n = 4). a.u., arbitrary units. *p<0.05; **p<0.005. The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s001" target="_blank">S2</a>.</p

A3V gene transduction in post-hatch chick brain.

<p>(A–C) EGFP expression 1 week after A3V injection (a total of 5×10⁷, 5×10⁸, and 5×10⁹ GC, respectively) was analyzed by immunofluorescence labeling of parasagittal sections. Arrowheads indicate injection sites in the striatum. Scale bars are 1 mm. (D) Gene transduction after LV or A3V injection was quantified by measurements of EGFP-expressing area in the parasagittal sections containing injection sites (n = 4). The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s004" target="_blank">Table S4</a>. (E) A3V-treated striatal cells were visualized by double immunofluorescence labeling for EGFP (green) and neuronal marker NeuN (red). Scale bar indicates 50 µm. (F) EGFP expression 24 hrs after A3V injection (a total of 5×10⁹ GC) into the intermediate medial mesopallium (IMM) was analyzed by immunofluorescence labeling of coronal sections. The arrowhead indicates the A3V injection site. Scale bar is 1 mm.</p

Neuron-preferential transduction of A3V vector.

<p>(A) Primary cultures of chick neural cells after A3V or LV infection were immuno-labeled with antibodies against EGFP (green) and the neuronal marker MAP2 (red). Arrowheads indicate MAP2-negative, LV-transduced cells. Scale bar indicates 20 µm. (B) The neuronal transduction rates of A3V and LV are represented as the percentage of MAP2 and EGFP double-positive cells within 100–200 EGFP-positive cells (n = 4). *p<0.005. The raw data are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048730#pone.0048730.s003" target="_blank">Table S3</a>.</p