Fig. 1
Virus infection on the whole explant.
a. Schematic diagram illustrating the model used in this protocol.
b. Scheme illustrating the schedule of the experiment.
c-d. The filter with 1.0 μm pore size shows higher rAAVs infection efficiency compared with 0.1 μm. The explants were incubated in culture medium containing 1.4 × 10
9 vg (virus genome)/ml AAV2-Cre (which harbors Cre recombinase) for 2 days. All the progenies of the infected cells will be labelled with tdTomato expression. The images were taken with stereomicroscope on day 2, 4 and 6 to monitor the growth of mammary buds and the expression of tdTomato reporter genes (
c). The exposure time of tdTomato expression has been labelled in the image. The virus infection efficiency has been further confirmed by whole mount fluorescent immunostaining with anti-EpCAM antibody and Hoechst 33342 to visualize the epithelial cells and nuclei, respectively, and imaged with Zeiss LSM700 confocal microscope and the representative images of optical section were shown (d).
e-f. Different serotype of rAAVs preferentially target different tissues. The explants were incubated in culture medium containing 8.6 × 10
9 vg/ml AAV2-Cre, AAV8-Cre or AAV9-Cre, respectively, for 2 days. 7 days after culture, the virus infection efficiency was further confirmed by whole mount fluorescent immunostaining and confocal microscope and the representative images of optical section were shown (
e). The infection efficiency in mammary epithelium and mesenchyme compartment from the same explant was determined separately by quantifying the progenies of infected and non-infected cells using Imaris 9.2 software (Bitplane). The figure was produced with R, a free software environment available at
http://www.r-project.org/, and ggplot2 package [
18] (
f). The data are presented as mean ± SD. Statistical comparisons were performed by paired Student’s t test