Transduction of terminally differentiated neurons by avian sarcoma virus

Abstract

Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.

FIG. 1.

Mouse embryonic hippocampal neuron explants. The hippocampus was isolated from BL/6 mouse embryos between days 14.5 and 16.5 of gestation. The hippocampus was stripped of meninges, and a single-cell suspension was plated. The neuronal progenitors divide once and become fully differentiated neurons after approximately 4 days in culture. Representative differential interference contrast micrographs are shown. Panels: A, neuronal progenitors 1 day after isolation; B, differentiated neurons after 5 days in culture; C, MAP2 staining of the same differentiated neurons that were fixed and incubated with an anti-MAP2 antibody (red). 4′,6′-Diamidino-2-phenylindole (DAPI) (DNA)-stained nuclei are shown in blue.

FIG. 2.

Transduction of neurons with an ASV-GFP vector. (A) Neuronal cultures were infected with the same virus stock, and GFP reporter expression was examined 3 days postinfection by microscopy. Representative fluorescent fields (GFP expression, left) and phase-contrast fields (right) are shown. Cultures of neural progenitors infected 1 day after isolation (top) and differentiated neurons infected 5 days after isolation (bottom) are shown. (B) ASV transduction correlates with integration detected by B2-PCR. Neurons were infected 5 days after isolation. Total cellular DNA was isolated 3 days postinfection. B2-PCRs of uninfected neurons, infected neurons, infected murine fibroblasts, and infected neuron DNA amplified without B2 primers are shown.

FIG. 3.

MAP2-positive neurons are transduced by the ASV reporter virus. Neurons were infected 5 days after isolation, and at 3 days postinfection they were fixed and stained for MAP2. MAP2 is localized to the dendrites and around the cell body, while GFP is localized throughout the cell. GFP-expressing cells were frequently MAP-2 positive. Representative fields of fluorescent staining by MAP2 (left) and GFP expression (center) and their overlay (right) are shown.

FIG. 4.

Assays for cell cycling. (A) BrdU pulse of dividing progenitor cells that were stained for BrdU after 2 days. Representative differential interference contrast (left) and fluorescence (right) fields are shown. (B) BrdU pulse of differentiated neurons. Differentiated neurons after 5 days in culture were treated as described above. (C) PCNA levels in dividing progenitor cells. Dividing progenitor cells cultured for 1 day were fixed and stained with an anti-PCNA antibody. (D) PCNA levels in differentiated neurons. Differentiated neurons were cultured for 5 days before they were fixed and stained with an anti-PCNA antibody.

FIG. 5.

Transduction efficiencies of ASV, HIV-1, and MLV. Dividing progenitor cells on the day of isolation and differentiated neurons after 5 days in culture were infected with the same stock of virus. The number of GFP-positive cells was determined by fluorescence-activated cell sorter analysis approximately 3 days postinfection. Transduction efficiency was determined by the ratio of GFP-expressing cells in the dividing progenitors to differentiated neurons. The mean transduction efficiency from at least two parallel experiments carried out in triplicate is shown with the standard deviation between the experiments.