Disruption of the endothelial nitric oxide synthase gene affects ovulation, fertilization and early embryo survival in a knockout mouse model.

Two consecutive experiments determined whether disruption of the endothelial nitric oxide synthases (NOS) gene (Nos3) affects ovulation, fertilization, implantation, and embryo development. In the first trial, Nos3-knockout mice (groups Nos3(-/-)) and wild-type mice (groups Nos3(+/+)) showed significant differences in mean number of corpora lutea (9.7+/-1.2 in Nos3(-/-) versus 14.2+/-1.2 in Nos3(+/+); P<0.01), rate of anovulation (48.3+/-7.3% in Nos3(-/-) versus 29.7+/-6.3 in Nos3(+/+); P<0.05), total mean number of recovered oocytes/zygotes (4.0+/-1.1 in Nos3(-/-) versus 10.4+/-1.6 in Nos3(+/+); P<0.01), and non-fertilization rate (50.7 in Nos3(-/-) versus 3.3% in Nos3(+/+); P<0.001). In the second trial, implantation and early pregnancy losses in Nos3-knockout and wild-type dams were detected by real-time ultrasound imaging. The number of embryos reaching implantation was higher in Nos3(+/+) than in Nos3(-/-) mice (7.5+/-0.4 vs 4.0+/-0.4; P<0.005); thereafter, embryo losses were detected between days 8.5 and 13.5, in 62.5% of the Nos3-knockout dams and, at days 10.5 and 11.5, in 16.7% of the control females (P<0.005). Thus, NO and NOS3 deficiencies affect reproductive and developmental features in the Nos3-knockout mouse model.

Dido gene expression alterations are implicated in the induction of hematological myeloid neoplasms.

The myelodysplastic/myeloproliferative diseases (MDS/MPDs) are a heterogeneous group of myeloid neoplasms that share characteristics with chronic myeloproliferative diseases and myelodysplastic syndromes. The broad spectrum of clinical manifestations makes MDS/MPDs extremely difficult to diagnose and treat, with a median survival time of 1-5 years. No single gene defect has been firmly associated with MDS/MPDs, and no animal models have been developed for these diseases. The association of deletions on chromosome 20q with myeloid malignancies suggests the presence of unidentified tumor suppressor genes in this region. Here we show that the recently identified death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing, and we map the human gene to the long arm of chromosome 20. We found that targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which we also found in other myeloid but not lymphoid neoplasms or in healthy donors. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment.

Increased gene dosage of Ink4a/Arf results in cancer resistance and normal aging.

Mammalian genes frequently present allelic variants that differ in their expression levels and that, in the case of tumor suppressor genes, can be of relevance for cancer susceptibility and aging. We report here the characterization of a novel mouse model with increased activity for the Ink4a and Arf tumor suppressors. We have generated a "super Ink4a/Arf" mouse strain carrying a transgenic copy of the entire Ink4a/Arf locus. Cells derived from super Ink4a/Arf mice have increased resistance to in vitro immortalization and oncogenic transformation. Importantly, super Ink4a/Arf mice manifest higher resistance to cancer compared to normal, nontransgenic, mice. Finally, super Ink4a/Arf mice have normal aging and lifespan. Together, these results indicate that modest increases in the activity of the Ink4a/Arf tumor suppressor result in a beneficial cancer-resistant phenotype without affecting normal viability or aging.

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background.

Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic-severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar "humanization" rationale may be required.

Genetic inactivation of Par4 results in hyperactivation of NF-kappaB and impairment of JNK and p38.

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF-kappaB pathway. This may explain its pro-apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4(-/-) mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor-alpha (TNF-alpha) to induce apoptosis by increased activation of NF-kappaB. Consistent with recent reports demonstrating the antagonistic actions of NF-kappaB and c-Jun amino-terminal kinase (JNK) signalling, we have found that Par4(-/-) cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF-alpha and interleukin 1. Higher levels of an anti-apoptotic JNK-inhibitor protein, X-chromosome-linked inhibitor of apoptosis, in Par4(-/-) EFs might explain the inhibition of JNK activation in these cells.

"Super p53" mice exhibit enhanced DNA damage response, are tumor resistant and age normally.

The tumor suppressor p53 is critical in preventing cancer due to its ability to trigger proliferation arrest and cell death upon the occurrence of a variety of stresses, most notably, DNA damage and oncogenic stress. Here, we report the generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes. Prior to this, we demonstrate that the p53 transgenic allele (p53-tg), when present in a p53-null genetic background, behaves as a functional replica of the endogenous gene. "Super p53" mice, carrying p53-tg alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage. Importantly, "super p53" mice are significantly protected from cancer when compared with normal mice. Finally, in contrast to previously reported mice with constitutively active p53, "super p53" mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control. Together, our results prove that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.