Figure 3.

OPN expression in embryonal cell lines and pregastrulation embryos. (A) Northern blot analyses of OPN mRNA (Opn) expression in F9 EC, GCLB, MBL-1 ES, and fibroblast (3T3) cell lines. Each lane contains 1 μg of poly(A)⁺ RNA. mRNA levels were normalized by comparison of corresponding ethidium bromide-stained 28S and 18S rRNA bands. (B) RT–PCR of OPN and Oct-4 mRNA in different stages of preimplantation embryos. Multiplex PCR of Oct-4 and Opn was performed on reverse transcripts obtained from F9 cells, morulae (Mo), blastocysts of 3.5 dpc (Bl 3.5) and 4.5 dpc (Bl 4.5). PCR negative controls were done with H₂O (PCR Co) and M2 medium treated with RNA extraction buffers and reverse transcription buffers (M Co). (C) In situ hybridization of 3.5–6.5 dpc embryos with OPN DIG-labeled mRNA. Blastocyst staining is observed in ICM and forming hypoblast, but not in trophectoderm and 4.5 dpc hypoblast. In sections of 5.5 and 6.5 dpc embryos only granulated metrial gland cells (GMGC) are stained. (E) embryo. Hybridization with sense probes did not generate staining (not shown).