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.