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Fig. 2. Basonuclin-RNAi transgene and its effect on female fertility, levels of
basonuclin mRNA and protein in oocytes. (A) Structures of the two
transgene constructs with different vector backbones, splicing sequences and
basonuclin cDNA sequences. The nucleotide coordinates of mouse basonuclin cDNA
are shown above one copy of the inverted repeats (black arrows).
(B,C) Fertility of the transgenic RNAi females was inferred by
the average litter-size. Transgenic females (founder, B, or out-crossed
progeny of the founder males, C) were mated with three normal males in a
randomized sequence. (B) The data are based on three matings for each
transgenic founder, and the control (non-TG) is the average litter size of the
seven non-transgenic females. (C) The mating/pregnancy number (n) is shown
above each bar. *P<0.001. Basonuclin mRNA was
knocked-down in transgenic GV-stage oocytes as shown by real-time PCR (D),
immunocytochemistry (F) and immunoblot (G). (D) The mRNA for the
upstream-binding factor (UBF), a ubiquitous Pol I transcription factor, was
used as a control. (E) Real-time PCR data were converted into the
percentage of wild-type basonuclin mRNA level and plotted against the
fertility data from C. (F) Oocytes were stained with anti-basonuclin
antibody (red) and DAPI (blue), and the two images are overlaid to indicate
the position of germinal vesicle in the transgenic oocytes. Scale bar: 10
µm. (G) Two duplicate samples were analyzed for each transgenic
line. Each lane contains protein extracted from 50 oocytes. After probing for
basonuclin, the membrane was stripped and re-probed with antibody against
ß-tubulin. TG, transgenic oocytes; non-TG, non-transgenic oocytes.
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