Background: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cremediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue.

Complementary sets of genes are epigenetically silenced in male and female gametes in a process termed genomic imprinting. The Dnmt3L gene is expressed during gametogenesis at stages where genomic imprints are established. Targeted disruption of Dnmt3L caused azoospermia in homozygous males, and heterozygous progeny of homozygous females died before midgestation. Bisulfite genomic sequencing of DNA from oocytes and embryos showed that removal of Dnmt3L prevented methylation of sequences that are normally maternally methylated. The defect was specific to imprinted regions, and global genome methylation levels were not affected. Lack of maternal methylation imprints in heterozygous embryos derived from homozygous mutant oocytes caused biallelic expression of genes that are normally expressed only from the allele of paternal origin. The key catalytic motifs characteristic of DNA cytosine methyltransferases have been lost from Dnmt3L, and the protein is more likely to act as a regulator of imprint establishment than as a DNA methyltransferase.

Although the hormone erythropoietin (Epo) and its receptor (EpoR) are known to play important roles in the regulation of erythropoiesis, several questions remain concerning the developmental role of Epo/EpoR signaling. As the functions of Epo have been defined primarily through studies of definitive erythroid cells, its importance for primitive, embryonic erythropoiesis remains uncertain, as does the significance of EpoR expression in several nonerythroid cell types. To address these questions, mouse embryonic stem cells and embryos lacking a functional EpoR gene were produced by gene targeting. The effects of the mutation were examined in embryos developing in vivo, in chimeric adult mice produced with homozygous mutant embryonic stem cells, and in hemopoietic cells cultured in vitro. No defects were apparent in nonerythroid cell lineages in which the EpoR normally is expressed, including megakaryocytes and endothelial cells. In the mutant yolk sac, primitive erythrocytes were produced in normal numbers, they underwent terminal differentiation, and expressed near normal levels of embryonic globins, although they were reduced in size and their proliferation was severely retarded after E9.5. In contrast, in the fetal liver, definitive erythropoiesis beyond the late progenitor (CFU-E) stage was drastically inhibited by the EpoR mutation, and virtually no definitive erythrocytes were produced in vivo, leading to embryonic death by E13.5. Thus, our results suggest a fundamental difference in the molecular mechanisms stimulating primitive and definitive erythropoiesis. It was also observed that a few mutant definitive erythroid cells could terminally differentiate when cultured with additional cytokines, demonstrating that although Epo/EpoR signaling is important for definitive erythroid cell survival and proliferation, it is not an obligatory step in differentiation.

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1−/− mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3 −/− and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, ␤-catenin, GSK3␤, and APC. Upon [Keywords: MACF1-knockout mice; Wnt signaling; Axin translocation; LRP5/6; embryonic lethality; primitive streak; mesoderm] Supplemental material is available at http://www.genesdev.org.

PKC-interacting protein (PKCI), also designated histidine triad nucleotide-binding protein 1, belongs to the histidine triad (HIT) family of proteins. Its structure is highly conserved from bacteria to humans and shares homology with the tumor-suppressor gene fragile histidine triad (FHIT). Although it was originally thought to inhibit PKC, its actual physiologic function is not known. Therefore, we used the technique of homologous recombination to generate homozygous deleted PKCI ؊/؊ mice. These mice display normal fetal and adult development. However, when mouse embryo fibroblasts were established from 13.5-day embryos and serially passaged the PKCI ؊/؊ cells displayed an increase in growth rate and underwent spontaneous immortalization, whereas the PKCI ؉/؉ cells senesced and ceased growing. Furthermore, the PKCI ؊/؊ mouse embryo fibroblasts displayed increased resistance to cytotoxicity by ionizing radiation. In view of these findings we examined possible effects of PKCI on susceptibility to carcinogenicity. Both PKCI ؉/؉ and PKCI ؊/؊ mice were treated with the chemical carcinogen N-nitrosomethylbenzylamine (NMBA) by intragastric administration and killed 12 weeks later. As expected with this protocol, NMBA induced squamous tumors (both papillomas and carcinomas) of the forestomach. The incidence, multiplicity per mouse, volume, and degree of malignancy of these tumors were significantly greater in the PKCI ؊/؊ than in the PKCI ؉/؉ mice. Furthermore, four adenomas and one adenocarcinoma of the glandular stomach were found in the NMBA-treated PKCI ؊/؊ mice but no tumors of the glandular stomach were found in the NMBA-treated PKCI ؉/؉ mice or in any of the untreated mice. Taken together, these findings suggest that, like FHIT, PKCI may normally play a tumorsuppressor role. The possible role of PKCI as a tumor suppressor in humans remains to be determined.tumor suppressor

Expression of a kinase-deficient ATM protein leads to severe genomic instability and embryonic lethality.

Loss of TGFBI, a secreted protein induced by transforming growth factor-b, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene-induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI À/À MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI À/À MEFs, which correlates with aberrant activation of transcription factor cyclic AMPresponsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI À/À cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI À/À mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo.

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca 2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration , were challenging to interpret because of negative effects on synaptic transmission (45). We therefore altered our approach to limit ablation of Sirt6 to rod photoreceptors with an inducible gene disruption strategy. Using this model, we tested whether upregulation of glycolytic flux through Sirt6 knockout can preserve both rod and cone photoreceptors in a preclinical, Pde6-associated RP model. The Journal of Clinical Investigation R E S E A R C H A R T I C L E ResultsGeneration of experimental and control groups. The third most common cause of autosomal recessive RP is deficiency in the PDE6 enzyme, which controls the depolarization state of rods by regulating cGMP levels (9, 46-48). An established preclinical model for RP involves a homozygous point mutation (H620Q) in the gene months by increasing glucose uptake and utilization for NADPH production in 4 different mouse models of RP (11,12). In our report, we propose a similar strategy that improves both survival and function of degenerating rods and cones. We hypothesized that Pde6-associated RP provokes a metabolic aberration in the rod cells that forces them to succumb to the consequences of elevated cGMP and Ca 2+ via cyclic nucleotide-gated (CNG) channels and Na + /Ca 2+ -K + exchangers (36-39). The histone deacetylase sirtuin 6 (SIRT6) is a transcriptional repressor of glycolytic enzymes that has been extensively studied in the context of metabolism and cancer biology (40). Normally, S...