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The scraped epithelium was immediately collected in a tube containing RNA extraction buffer, Trizol (Invitrogen) and extraction proceeded according to manufacturer's recommendations. Equal amounts of extracted RNA from BIBW2992 mice per group were pooled for cDNA synthesis. cDNA synthesis was done using RT2 First Strand kit (SABiosciences) according to manufacturer's recommendation. Real-Time PCR array was done using RT2 Profiler PCR Array (SABiosciences) for mouse Wnt signaling pathway (cat. #PAMM-043A-2) or ready-made individual primers encoding regulatory genes of differentiation (QuantiTect Primer Assay, Qiagen). Samples were run in triplicates on Real-time thermal cycler Mastercycler? ep realplex machine (Eppendorf). To delete Klf4 from the intestine, we employed Vil/Cre mice, which carry the Cre recombinase directed by 12.4?kb of the mouse villin promoter ( Madison et al., 2002). Villin is normally expressed in the gut epithelium and the proximal tubule of the kidney beginning at embryonic day 11 ( Maunoury et al., 1988?and?Maunoury et al., 1992). Vil/Cre mice were crossed with mice carrying floxed alleles of Klf4 (Klf4fl/fl) ( Katz et al., 2005) and then backcrossed to obtain Vil/Cre;Klf4fl/fl (Klf4?IS) mice. Klf4fl/fl mice without Cre served as controls. Klf4��IS mice were born in a Mendelian ratio, and at three weeks of age appeared grossly normal without any significant difference in weight from the control Klf4fl/fl or wild type mice. Deletion of the Klf4 gene from the intestine was confirmed by immunohistochemistry. Klf4 staining was seen in the differentiated intestinal epithelial NVP-BKM120 manufacturer SWAP70 cells of both the small and large intestines of the control mice ( Fig. 1A and C), while absent from the epithelia of the small and large intestines of the Klf4��IS mice ( Fig. 1B and D), confirming Cre-mediated deletion of Klf4 from the intestinal epithelium. Macroscopically, both the small and large intestines of the Klf4��IS mice appeared normal. At 3?weeks of age, histology of the small intestinal epithelium of Klf4��IS mice appeared normal ( Fig.?1F) as compared to controls ( Fig.?1E). In contrast, the colonic epithelium of the Klf4��IS mice had a distorted architecture with increased glandular formation and reduced numbers of goblet cells (compare Fig. 1G and H). No evidence of hypertrophy or hyperplasia was observed in either the small or large intestine of the Klf4��IS mice up to 10?months of age. Since KLF4 negatively regulates cellular proliferation, we examined the effect of Klf4 deletion on the rate of proliferation of intestinal epithelial cells in vivo. At 3?weeks of age, mice were pulse-labeled in the S-phase with bromodeoxyuridine (BrdU) and sacrificed at 4 and 24?h later for immunohistochemical examination. The number of BrdU-positive cells was higher in the small intestine of Klf4��IS mice compared to the control littermates at both 4?h (p?