RNAi of Grp78 may disturb the fusion of ICR mouse palate cultured in vitro

Abstract

RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.