Figure 1.
Generation of Alpaca organotypic culture.
A) Representative phase contrast image (10x) of an Alpaca skin organoid, delimited by a yellow dotted line, with sprouting cells as indicated by yellow arrows. B) Representative phase contrast image (20x) of the first passage primary cell culture obtained from the cells sprouting from the organoids. C) Representative phase contrast images (10x) of three independent clones of cells after immortalization with SV40 large T antigen.
Figure 2.
A) Exponential regression curve by which ASSCs doubling time (DT) and growth rate (GR) was calculated. B) SV40 large and small T antigen expression in ASSCs at different passages (5, 10, 15, 20, 30, 40 and 60) as monitored by Western immunoblotting. A cross reactive protein band (CB) is detected also in the negative control (−), ASSC primary culture. The ladder lane is indicated by Ld. C) Phase contrast and fluorescent images (20x) of ASSCs from the 60th passage indicating expression of the stromal marker, vimentin. Counterstained nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) were merged with green fluorescent image (merge). ASSCs from the 3rd, 10th, and 20th passage stained similarly with α-vimentin antibody (not shown).
Figure 3.
Representative phase contrast images (10x) of uninfected and infected (CpHV-1, BoHV-1 and BVDV) ASSCs 48 h post infection, along with the replication kinetics of the respective viruses expressed as log TCID50/ml. The data presented are the means ± standard errors of triplicate measurements (P>0.05 for all time points as measured by Student’s t test).
Figure 4.
Neospora caninum infection of ASSCs.
Representative phase contrast image (20x) of ASSCs containing Neospora caninum tachyzoites and cell free Neospora caninum tachyzoites at 48 h post infection.
Figure 5.
Representative phase contrast and fluorescence images (10x) of ASSCs transfected with a GFP expression plasmid by different methods (Electroporation, LTX, PEI and Fugene HD), along with the efficiency of transfection as measured by cytometry. Each set of transfections was performed three times and standard deviations were negligible.
Figure 6.
Representative phase contrast and fluorescence images (10x) of ASSCs transduced by a self-inactivating replicating incompetent third generation lentiviral vector at 48 h post transduction (A) and a replicating competent BoHV-4-based vector (B) at different times: 24 and 48 h post infection (P.I.). Efficiency of transduction was measured by cytometry. The titres of BoHV-4-based vector were examined by determining the numbers of cell free viruses through time (expressed as log TCID50/ml). The data presented are the means ± standard errors of triplicate measurements (P>0.05 for all time points as measured by Student’s t test).
Figure 7.
A) Representative in vivo bioluminescence image of ASSCs transduced with different doses (1 and 0.1 transducing units (T.U.)/cell) of a lentiviral vector expressing luciferase. B) Representative in vivo bioluminescence image of FVB mice displaying ASSCs transduced with a lentiviral vector expressing luciferase and localized to the lung 4 h post injection.
Figure 8.
A) Representative in vivo bioluminescence images of nude mice subcutaneously inoculated with 3×106 lentiviral transduced ASSCs on one side and 106 on the opposite side of their body. Luminescence signal was acquired weekly until it was no longer detectable (B).