The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in ≈60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

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Changes in biochemical markers of bone turnover following intermittent injections of human (h)PTH (1-34) suggest that bone formation is initially favored over bone resorption. hPTH (1-34) is also known to influence osteoclast maturation and activity through modulation of osteoblast-derived cytokines, such as receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), IL-6, and IL-6 soluble receptor (IL-6sR). In this experiment, we investigated the changes in serum levels of soluble RANKL (sRANKL), OPG, IL-6, and IL-6sR in patients with glucocorticoid-induced osteoporosis treated with hPTH (1-34). Fifty-one postmenopausal women with glucocorticoid-induced osteoporosis were randomized to receive 12 months of 400 U hPTH (1-34) ( approximately 40 microg) daily and standard hormone replacement therapy, or hormone replacement therapy alone. Serum levels of sRANKL, OPG, IL-6, and IL-6sR were measured at baseline, 1 month, and every 3 months thereafter for a total of 24 months. hPTH (1-34) caused a rapid and significant increase in sRANKL within 1 month, and the levels remained elevated throughout the duration of therapy. IL-6 and IL-6sR increased significantly within 1 month, but returned to baseline levels more rapidly. In contrast, OPG was mildly suppressed beginning 6 months after hPTH therapy. These data support the hypothesis that hPTH (1-34) initially stimulates osteoblast maturation and function, which in turn leads to osteoclast activation and a gradual rebalancing of bone formation and resorption.

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