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Fig. 7. Effects of transitin knockdown by shRNA on neuroepithelium cell
fate. (A) Transfection with pSilencer-Trans289 shRNA
expression vectors successfully knockdowns transitin expression. At 24 hours
after electroporation the transfected area, revealed by the expression of
co-transfected EGFP, shows weak expression of transitin (purple;
arrowheads). (B) Trans289-transfection results in a
downregulation of Numb expression (purple). The reduction of anti-Numb
immunoreactivity is evident in the basal processes of neuroepithelium (NE)
cells. (C) BrdU uptake of neural tube cells co-transfected with
EGFP and pSilencer-Trans289. Little overlap of
GFP-fluorescence and anti-BrdU immunostaining is observed. (D) The
proportion of BrdU-GFP double-positive neural tube cells co-transfected with
EGFP and empty pSilencer (-; green), or pSilencer carrying mutated
transitin sequence (Trans289m, mut; blue), or pSilencer carrying the
wild-type transitin sequence (Trans289, wt; red), 24 hours after
transfection. (E) Proportion of TUNEL-GFP double-positive neural tube
cells co-transfected with EGFP and pSilencer (-), Trans289m
(mut), or Trans289 (wt) 24 and 48 hours after transfection.
(F) Neuronal differentiation of transfected neural tube cells
co-transfected with EGFP and Trans289 (wt) 48 hours after
transfection. Whereas most empty pSilencer-transfected cells remain
Hu-negative in the ventricular zone (left panel),
Trans289-transfected cells migrate basally, express Hu and
intermingle with untransfected neurons (right panel). (G) The
proportion of Hu-EGFP double-positive neural tube cells co-transfected with
EGFP and pSilencer (-), Trans289m (mut), or
Trans289 (wt) 24 and 48 hours after transfection. Five embryos
(approximately 200-300 cells/embryo) were examined to obtain each bar in
D,E,G. Error bars indicate standard deviations.
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