Fig 1.

Bovine SETDB1 expression and siRNA mediated depletion in bovine preimplantation embryos. A) Measurements of transcripts encoding bovine SETDB1 during embryogenesis. Levels of SETDB1 mRNAs were measured in bovine oocytes and at various stages of preimplantation development using q-RT-PCR. B) Validation of siRNAs targeting SETDB1 using transient transfection into bovine fetal fibroblasts. SETDB1 transcript levels were quantified using q-RT-PCR. C–D) Measurement of SETDB1 transcripts in bovine 8-cell (C) and morula stage embryos (D) from the Control, siNull and siSETDB1 treatment groups using q-RT-PCR. For A–D, samples were normalized to the geometric mean of GAPDH, YWHAZ & SDHA and graphed using the 2^{−ΔΔ CT} method [52]. Graphs are representative of four independent experiments. Error bars represent the SEM, *** denotes p < 0.001 and **** p < 0.0001. E–G) Developmental arrest of siSETDB1 treated embryos. Confocal images of expanded blastocyst stage noninjected Control (E), and siNull embryos (F), as well as arrested morula stage siSETDB1 injected embryos (G). 20x images displaying the injected green fluorescent dextran dye are displayed on the right while bright field images are displayed on the left.