Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell * tetraploid embryo aggregation. One line, designated Rl, produced live offspring which were completely ES cellderived as judged by isoenzyme analysis and coat color. These cell culture-derived anhnas were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of Rl, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the oiginal totipotency and gave rise to viable, completely ES cell-derived animals. The (Nunc). Most embryos hatched and attached to the feeders by day 2 after plating. The inner cell masses (ICMs) were left to grow for 4 more days, when they were mechanically disaggregated in their own wells by using drawn-out Pasteur pipettes. Four to five days later, the cells were transferred into new wells either by trypsinizing or by mechanically disaggregating the undifferentiated colonies. We started counting passage number when we were first able to pass the cells into 35-mm plates (passage 1). The cells were first frozen at passage 5, which was "'3 weeks after the blastocyst stage. Four cell lines were established, designated Rl, R2, R6, and R13.Production of Tetraploid Embryos. The oviducts of superovulated and mated CD1 (GPI-BB) females were flushed 44-46 hr after treatment with human chorionic gonadotropin to collect late two-cell-stage embryos. The embryos were placed one at a time between two platinum electrodes laid 250 ,um apart in M2 medium (7) in the electrode chamber (8). The blastomeres were fused by a short electric pulse (9)

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.