One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3 -untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1 ؊/؊ embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1 ؊/؊ embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.

The Wnt/b-catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45-mutated-b-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated b-catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin-D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2-fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S-phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of b-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of b-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, b-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. b-Catenin-overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed upregulation of b-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: b-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, b-catenin overexpression makes TG mice susceptible to DEN-induced HCC. (HEPATOLOGY 2010;51:1603-1613 W nt/b-catenin signaling is an evolutionarily well-conserved pathway and important in liver health and repair.1 In adult liver, bcatenin signaling is essentially quiescent, with active bcatenin restricted to hepatocytes in the centrizonal area where it regulates expression of genes such as glutamine synthetase (GS) and others involved in xenobiotic metabolism.2 In other hepatocytes, b-catenin steady state is achieved by phosphorylation at key serine/threonine residues and subsequent degradation, and is predominantly localized to membrane to mediate cell-cell adhesion by forming a bridge between E-cadherin and actin cytoskeleton. Activation of b-catenin signaling during liver regeneration has been reported in rats and mice. [4][5][6][7] Although a positive regulator in the activity of normal liver growth, aberrant activation of the Wnt/bcatenin pathway is implicated in hepatocarcinogenesis,

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine aA-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and aA-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the aA-CAT fusion gene and the endogenous aA-crystallin gene are coregulated during lens development in the transgenic mice. These experiments show that the murine aA-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.