The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse IntroductionColony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells and is the primary regulator of mononuclear phagocyte production in vivo. 1,2 However, CSF-1 also regulates cells of the female reproductive tract and plays an important role in fertility. 3,4 The effects of CSF-1 are mediated by a high-affinity receptor tyrosine kinase (CSF-1R) [5][6][7][8] encoded by the c-fms proto-oncogene. 9 The CSF-1R is expressed on primitive multipotent hematopoietic cells, 10,11 mononuclear phagocyte progenitor cells, 12 monoblasts, promonocytes, monocytes, 5,6 tissue macrophages, 6,13-15 osteoclasts, 16 B cells, 17,18 smooth muscle cells, 19 and neurons. 20,21 CSF-1R messenger RNA (mRNA) is expressed in Langerhans cells, 22 in the female reproductive tract, in oocytes and embryonic cells of the inner cell mass and trophectoderm, 23 in decidual cells, [24][25][26] and in cells of the trophoblast. 24,25 The expression of the CSF-1R on primitive hematopoietic cells that are unable to proliferate in vitro in response to CSF-1 alone 10,11 but are able to proliferate and differentiate if stimulated with combinations of CSF-1 and other hematopoietic growth factors 10,11,27 suggests that CSF-1R is involved in the regulation of more primitive hematopoietic cells than those that form macrophage colonies in vitro in response to CSF-1 alone.Mice homozygous for the mutation osteopetrotic 28 possess an inactivating mutation in the coding region of the CSF-1 gene and are devoid of detectable CSF-1. 29,30 These Csf1 op /Csf1 op mice are osteopetrotic because of an early and marked deficiency of osteoclasts 28 that spontaneously recovers with age, 31,32 probably because of the action of vascular endothelial growth factor. 33 However, the phenotype of these mice is pleiotropic. 3 They are toothless; have low body weight, low growth rate, and skeletal abnormalities; and are deficient in tissue macrophages. 2,28,30,34,35 They have defects in both male and female fertility, neural development, the dermis, and synovial membranes. 3 The pleiotropic phenotype of the Csf1 op /Csf1 op mouse may be due to a reduction in trophic and/or scavenger functions of the tissue macrophages regulated by CSF-1, secondary to the reduction of their concentration in tissues, 2 because outside the female reproductive tract the CSF-1R is primarily expressed in mononuclear phagocytes. 1,3 However, it is possible that some of these effects may also be due to loss of function of other cells such as neuronal cells and muscle precursors, which have also been reported to express the CSF-1R. 20,36 To address the questions of whether CSF-1 activates other receptors besides the CSF-1R and, conversely, whether the CSF-1R me...

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro-and anti-angiogenic therapies.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.

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A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laserassisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.Perhaps the most efficient way to elucidate gene function is by engineering gene mutations in ES cells and from these cells deriving mice that contain these genetic changes [1][2][3][4] . Mutant phenotypes in these genetically altered mice often provide important insights into gene function. Technological advances in producing genetic alterations in ES cells have resulted in large collections of modified ES cells 5-8 that will soon include mutations for every gene in the genome 9,10 . These advances have shifted the bottleneck in gene function assignment to the time-consuming steps involved in generating mutant mice that can be phenotyped. Currently, mutant mice are usually generated by introducing genetically altered ES cells into blastocyst-stage host embryos, resulting in F0 generation chimeric mice that are only partially derived from the modified ES cells. If part of the germ line is derived from the modified ES cells, these chimeras can be bred to obtain F1 generation mice that are uniformly heterozygous for the mutation of interest. Subsequent interbreeding of these heterozygous mice can result in F2 generation mice that are homozygous for the intended mutation. Because few mutant phenotypes can be detected in chimeric or heterozygous mutant mice, phenotyping requires derivation of homozygous mutant F2 mice. In addition, chimeras that are estimated to be 490% ES cell-derived based on coat color can be inefficient germline transmitters, because coat color chimerism does not fully reflect ES cell contribution to internal organs (including germ cells). In conclusion, chimeras render the production of homozygous mutant mice suitable for phenotyping inefficient and time-consuming.Use of tetraploid embryos as hosts for the ES cells can accelerate phenotyping because the resulting F0 generation mice are almost completely ES cell-derived and thus immediately available for phenotypic analyses [11][12][13] . Unfortunately, the tetraploid approach is an inefficient process for the generatio...