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Figure 3


Fig. 3. Morpholino oligonucleotides (MOs) targeted to the FGF8 mRNA. (A) Schematic of FGF8 gene diagramming the position of the MOs (red); FGF8b-specific alternatively spliced region (dark blue). (B) RT-PCR analysis of explants from embryos injected as indicated above the lanes. EF1{alpha}, loading control; explants were examined for xbra expression. F8b (FGF8b with the UTRs) (200 pg) induces xbra in explants (lane 4); co-injection of 40 ng of XlMOF8 effectively inhibits this effect (lane 6); xbra expression is rescued with injection of FGF8b-cds (does not have the MOF8 target sequence) (lane 7); FGF4 induction of xbra expression is unaffected by the MOF8 (lane 8). (C) XlMOF8 was designed to bind the translational start region of X. laevis FGF8. (D) Nucleotide sequence that MOSAF8a and MOSDF8 bind, respectively; MO sequence is in red. (E) Schematic of MOSDF8 and MOSAF8a effects on splicing. (F) RTPCR of X. laevis whole embryos injected as indicated; MOSAF8a (160, 120, 80 ng); MOSDF8 (170, 85, 43 ng); red brackets indicate MOSDF8 induced alternative splicing products that lead to premature termination; MOSAF8a results in a loss of the FGF8a but not FGF8b spliceform. (G) RTPCR of X. tropicalis embryos demonstrating the efficacy of MOSAF8a (32, 16 ng) and MOSDF8 (68, 34, 17 ng).