(c). mucin CYS domain, we generated three Tg founders that secreted into the intestinal lumen a string of 12 consecutive CYS domains. We chose the trefoil factor 3 (= 12) and Tg (= 11) mice was studied by confocal microscopy, which showed that the Tg mucus was less penetrable. The expression of the transgene was investigated by western blotting, immunohistochemistry (IHC), and fluorescence microscopy on fresh tissues. To analyze the secretion of the transgene into the Lotilaner mucus gel of the colon, we performed double staining for GFP and Muc2 using the agglutinin 1 (UEA1) lectin. The transgene, which was expressed in goblet cells, appeared to be widespread within the Muc2 gel layers at the cell surface of the colonic epithelium, as shown by IHC (Fig. 1b & c). Western blotting using an antibody directed against the CYS domain showed that the Tg222 recombinant molecule was secreted in the colon with a MW of ~170?kDa, which was in agreement with the expected size of the Tg product (Suppl. Fig. 1a). The profile of the expression of the transgene was then analyzed by IHC and fluorescence microscopy. Its tissue expression was similar to that of the gene24, with a high expression detected in salivary glands, intestine (goblet cells), and gallbladder (Suppl. Fig. 1b). The expression of the transgene was easily observed in non-fixed colon (Suppl. Fig. 1c), ileum, and gallbladder (data not shown) under epifluorescence microscope. Transgene expression was also observed in the colon using fiber-optic endomicroscopy on anesthetized mice and on fresh ex-vivo gallbladders (Suppl. Movies 1 & 2), as well as in the ileum (data not shown). These data indicate that our Tg lines secreted a recombinant molecule consisting of 12 copies of the MUC5B CYS domain #4 into the lumen of the intestine and that the Tg molecule became tangled within the Muc2 mesh of the mucus gel. The mucus gel, Muc2, and goblet cells are modified The main goal of this study was to modify the mucus layer to reinforce the mucus barrier. We began our analysis by examining more closely Muc2 expression via IHC. The mucus layer was thicker and/or more easily preserved in the colon of the two Tg lines (Tg208 and Tg222) than it was Rabbit Polyclonal to KLRC1 in WT mice, in which the mucus gel was barely visible (Fig. 1c). This suggests that the mucus is likely less friable after expression of the transgene. We then assessed whether the mucus properties differed between WT and Tg mice. Microspheres with the size of bacteria were added to the apical surface of colonic explants. After 45?min of sedimentation into the mucus, their distribution was studied. The relative proportion of beads was higher close Lotilaner to the epithelium in WT mice compared with Tg mice (Fig. 1d), which supported the notion that the transgene product is associated with a lower penetrability of the mucus blanket. In IHC experiments, we always observed a delocalization of the Muc2 immunostaining from the goblet cells in WT mice to the conserved mucus gel layers in Tg mice. The difference of immunostaining between WT and Tg mice with the Muc2 antibody may be explained by the different forms of glycosylation of Muc2 or interaction of Muc2 with the transgene product, masking the Muc2 antigenic epitopes. To assess the expression of mucin genes, semiquantitative polymerase chain reaction (PCR; TaqMan) was performed using colonic cDNA. We did not find any significant Lotilaner differences in or expression levels between Tg and WT mice (Suppl. Fig. 2a); is weakly expressed in the human terminal ileum and right colon25 and in the mouse duodenum and ileum26. To show the specificity of mucin CYS domains for the modification of secreted mucins, we determined the relative expression levels of (ortholog of the human gene27,28) and transcripts, the three main membrane-bound mucins expressed in colon, and no difference was found between Tg and WT mice (Suppl. Fig. 2b). Goblet cells were still present and filled with Alcian blueCperiodic acid Schiff (ABCPAS) material (Fig. 2a). No obvious difference in the number of goblet cells in the colon was detected between Tg and WT mice; however, there was a significant.