Tissue uptake of EGF receptor antisense oligonucleotides in organ culture of fetal mouse palates and their effects on in vitro palatogenesis

To investigate the incorporation of oligonucleotides (ODNs) into the tissues of cultured fetal mouse palates and their effects on in vitro palatogenesis, we cultured day-12.5 fetal mouse palates in a chemically defined serumless medium supplemented with either antisense or sense ODNs to epidermal growth factor receptor (EGF-r). The EGF-r ODNs were found to be incorporated into the palatal tissue and remained detectable for at least 72 hr. Immunohistochemical and immunoblot analyses revealed that the treatment with 5μM EGF-r antisense ODN suppressed the production of EGF-r protein. No pathological change was observed in the explanted palates when they were treated with 5 μM EGF-r antisense or sense ODNs, but the treatment with 10 or 20 μM ODN caused pyknotic changes in the palatal epithelium, probably due to the ODN toxicity. The present results indicate that under optimal conditions, antisense ODNs to EGF-r can be incorporated into fetal organs cultured in vitro and specifically inhibit the production of EGF-r protein. Since the suppression of the production of EGF-r protein did not prevent the palate fusion, EGF and/or EGF-r alone may not play a critical role in palatogenesis, as suggested by previous studies. The antisense ODN technique could be of potential use for analyzing the roles of specific molecules in normal and abnormal morphogenesis.