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Fig. 5. Morpholino oligonucleotides lipofection in the retina. (A) Retinal
lipofection of GFP Mo interferes with GFP translation. Embryos were lipofected
at stage 18 with GFP and a control Mo plus CD2, or with GFP and GFP Mo plus
CD2. In stage 41 embryos, the intensity of GFP fluorescence in retinal cells
was analysed. We used a filter to decrease the fluorescence light of the
microscope so that low GFP fluorescent cells are below the detectable
threshold. We then counted the number of lipofected retinas (CD2 positive)
that also contain GFP-positive cells. While 79% (n=43 retinas) of
CD2-positive retinas also contained GFP-positive cells when lipofected with a
control Mo, only 19% (n=42 retinas) contained GFP-positive cells when
lipofected with GFP Mo. (B) Xseb4R Mo block the translation of
Xseb4R mRNA. Synthetic Xseb4R mRNA (containing the
complementary sequence of Mo1) was injected into two-cell stage embryos
together with Xseb4R Mo1 or control Mo. Stage 10 embryo lysates were
then analysed by western blotting with a polyclonal anti-XSEB4R antibody and a
monoclonal anti-
tubulin antibody (control). In the presence of control
Mo, the anti-XSEB4R antibody recognises the 22 kD XSEB4 protein. The presence
of Xseb4R Mo1 specifically abolishes Xseb4R translation. (C)
Xseb4R loss-of-function delays differentiation of retinal cells. The
proportion of retinal cell types observed in retinas co-lipofected with GFP
plus a control Mo, GFP plus Xseb4R Mo1, or GFP plus Xseb4R
Mo2 was determined. Both Mo give the same results. The statistical analysis
was performed using the Student's t-test. *,
P<0.05; **, P<0.001; ***,
P<0.0001.
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