To specifically drive a liver-specific expression, the pWhere vec

To specifically drive a liver-specific expression, the pWhere vector was modified by inserting a regulatory element that consisted of the liver-specific alpha1-antitrypsin (α1-AT) promoter, coupled with the enhancer II (EII) sequence of human hepatitis B virus (HBV). This chimeric DNA element was previously shown to act as a potent, steady promoter and was able to ensure a constant, high FK506 research buy level of gene expression in the liver.20 The tissue specificity of this EII/α1-AT chimeric promoter, cloned upstream of a luciferase reporter gene into a pGL3 plasmid,

was tested in different types of hepatic and nonhepatic cell lines, which confirmed that the highest level of luciferase expression was detectable in hepatocytes, thereby

confirming the liver specificity of the promoter (data not shown). A DNA segment, which included the mmu-mir-221 locus, was amplified from mouse genomic DNA and was cloned into the pWhere/EII/α1-AT vector downstream of the EII/α1-AT promoter (Fig. 1A). Expression of miR-221 from this vector was proven to be functional in a liver-cancer–derived cell line (Supporting Fig. 1). To generate a line of TG mice, the pWhere/EII/α1-AT/miR221 plasmid was linearized using the PacI restriction enzyme. The purified 9-kilobase fragment containing the transgene was used to microinject fertilized oocytes of a B6D2F2 mouse strain to complete their development. After several crosses, a homozygous line of Selleck CP673451 TG mice overexpressing the miR-221 in the liver was produced and used in all subsequent experiments. To assess miR-221 expression levels in the TG model, livers taken from homozygous mice at different ages were analyzed by real-time polymerase chain reaction (PCR). In comparison with wild-type (WT) mice, the analysis revealed a stable, increased expression of miR-221 in the livers of TG animals, thereby confirming the development of homozygous

TG mice overexpressing miR-221 in hepatic cells (Fig. 1B). Macroscopically, livers of TG mice exhibited an increase in volume and weigth medchemexpress in comparison with controls (Supporting Fig. 2). Histologically, though both groups displayed a conserved liver architecture, TG livers were characterized by variable extents of steatohepatitic changes, with hepatocyte degeneration characterized by enlarged cells with large dysplastic nuclei, lipidic vacuole, and focal coagulative necrosis (Fig. 1C-F). These changes were more evident in older TG animals and were absent among WT controls. To assess whether miR-221 up-regulation could affect the expression of its targets, we performed an immunoblotting analysis to verify the expression of the miR-221 target proteins, Cdkn1b/p27, Cdkn1c/p57, and Bmf.2, 14 In non-neoplastic liver tissue, we confirmed that Bmf and Cdkn1b/p27 were both significantly down-regulated in TG mice. Cdkn1c/p57 was also generally down-regulated, although it did not reach statistical significance (Fig. 2).

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