Vascular endothelial growth factor (VEGF) and β‐catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone‐related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over‐expression in osteo‐chondroprogenitors and their progeny largely pheno‐copied constitutive β‐catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2‐ and phosphatidyl inositol 3‐kinase‐mediated pathway inducing β‐catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3‐β phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate β‐catenin activity may have widespread physiological and clinical ramifications.

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.

Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or E-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.The p53 tumor suppressor pathway is altered in virtually all human cancers (26). While the p53 gene itself is mutated or deleted in roughly 50% of cases, other mechanisms that disable p53 function are selected for in tumors with wild-type p53. Overexpression of MDM2, a critical negative regulator of p53, represents one way by which tumor cells escape p53-mediated tumor suppression (49). MDM2 inhibits p53 mainly, if not exclusively, by promoting its proteasome-dependent degradation through its E3 ubiquitin ligase activity (17,19,24,29).MDM2 is overexpressed in one third of all human sarcomas (37), 10% of glioblastomas and astrocytomas (41), 40% of oral squamous cell carcinomas (30), and 53% of myeloid (4) and 28% of B-cell (56) lymphomas. In addition, high levels of MDM2 are associated with poor prognosis in patients with non-Hodgkin's lymphoma (33). It is believed that overexpression of MDM2 is, at least in part, a direct consequence of gene amplification (34). However, high MDM2 levels have also been seen in many tumors that do not harbor gene amplification, indicating that there are additional mechanisms leading to MDM2 overexpression in human tumors.Ubiquitous overexpression of an Mdm2 transgene, under the control of its own promoter and regulatory regions, predisposes mice to spontaneous tumor formation (21). Tissuespecific Mdm2 overexpression in the basal layer of the epidermis induces hyperplasia and predisposes the epidermis to the formation of premalignant lesions and squamous cell carcinomas (11). These data establish a clear causal link between Mdm2 overexpression and increased tumor susceptibility in vivo. However, this effect is not clear-cut in all tissues: overexpression of Mdm2 in the granular layer of the epidermis does not promote tumorigenesis but rather affects its differentiation in a p...

To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/ loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf 164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our IntroductionErythrocytes or red blood cells (RBCs) fulfill the essential functions of facilitating gas exchange in the lungs and meeting tissue oxygen demands. They are continuously renewed from undifferentiated, selfgenerating cells throughout life in a tightly controlled growth process termed erythropoiesis. This process is part of the larger hematopoietic program that is defined by the production of all blood cells from hematopoietic stem cells (HSCs). These cells originate from different embryonic sites during development and are generated in close association with the vascular endothelium. The first hematopoietic cells detected during mouse embryonic development are the primitive erythroid cells of the yolk sac (YS) that are produced from precursors present in this tissue between embryonic days E7.0 and E8.25 and enter the circulation at E9.0. The definitive progenitors, that contain cells from the YS and the aorta-gonado-mesonephros (AGM) region, 1 reach the liver at E10.5 and give rise to the first definitive progenitors which subsequently colonize the bone marrow and spleen in the adult mouse. 2 Vascular endothelial growth factor (Vegf) is a secreted growth factor that mediates its biologic effects predominately by binding to either one of two transmembrane tyrosine kinase receptors, Vegfr-1(Flt1) and Vegfr-2(Flk1 or Kdr) as well as to the coreceptor Neuropilin1 (Nrp1). VEGF/VEGFR signaling pathways are essential for vascular and hematopoietic cell development. Indeed, heterozygous Vegf (ϩ/Ϫ) deficient embryos are devoid of most endothelial and hematopoietic cells in the blood islands and die around E10 as a consequence of severe cardiovascular abnormalities. 3,4 Gain of function studies showed that ubiquitous inducible expression of VEGF during early development leads to a paucity of mature RBCs. 5 During erythropoiesis in the developing YS Vegf is expressed in the YS mesoderm and extraembryonic visceral endoderm. 6 Using diploid and tetraploid embryo complementation approaches in conjunction with a Vegf hypomorphic all...

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