Summary: Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages.

C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight-cell-stage diploid embryo, we stably generated germline-competent ES-derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum- and feeder-free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background. genesis 47:414–422, 2009. © 2009 Wiley-Liss, Inc.

Several malignant tumor cells become apoptotic and revert to the benign phenotype upon parvovirus infection. Recently, we demonstrated that the rat parvovirus RPV/UT also induces apoptosis in the rat thymic lymphoma cell line C58(NT)D. However, a minority of cells that escaped apoptosis showed properties different from the parental cells, such as resistance to apoptosis, enhanced cell adherence, and suppressed tumorigenicity. The present study was performed to determine the molecular mechanism of parvovirus-induced phenotypic modification, including oncosuppression. We demonstrated that the nonstructural (NS) proteins of RPV/UT induced apoptosis in C58(NT)D cells and suppressed tumor growth in vivo. Interestingly, NS proteins induced the expression of ciliary neurotrophic factor receptor alpha, which is up-regulated in revertant cell clones, and enhanced histone acetylation of its gene. These results indicate that parvoviral NS regulate host gene expression through histone acetylation, suggesting a possible mechanism of oncosuppression.Autonomous parvoviruses are small, nonenveloped, nucleusreplicating viruses that contain a linear, single-stranded DNA genome of approximately 5 kb. The viral early and late promoters P4 and P38 encode the nonstructural proteins (NS-1 and NS-2) and the viral capsid proteins (VP-1 and VP-2), respectively (1). NS-1 is essential for viral replication and lytic infection and has multifunctional properties, such as ATPase activity, DNA helicase activity, and transactivation of the P38 promoter (10,16,33,43). NS-2 is generated by alternative splicing and is expected to be correlated with translation of viral mRNA and capsid protein assembly through interaction with the nuclear export factor chromosomal region maintenance 1 (2,26,29). NS proteins show high sequence homology among parvoviruses and are considered to be correlated with cytotoxicity in permissive cells and parvoviral pathogenicity (28,35). Several autonomous parvoviruses, including H-1, Kilham rat virus, and minute virus of mice, have tumor-suppressing properties in vivo and in vitro (34). Recently, we showed that most cells of the malignant thymic lymphoma cell line C58(NT)D infected with an isolate of rat parvovirus (RPV/UT) underwent apoptotic cell death, while the revertant cell clone C58(NT)D/R derived from a minor population of cells that survived after virus propagation showed several different phenotypes and reduced tumorigenicity (40). Furthermore, modified phenotypes of C58(NT)D/R were maintained at least over 15 cell passages. These findings suggest that these parvoviruses may be promising candidate vectors for tumor gene therapy (3, 7), although the mechanism of parvovirus-induced oncosuppression is still unclear.DNA methylation and histone modifications, such as deacetylation and methylation of specific lysine residues at the N termini of histones H3 and H4, play key roles in epigenetic gene regulation, resulting in inappropriate expression or silencing of genes without corresponding changes in their DNA sequenc...