A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

Developmentally regulated expression of mouse HtrA3 and its role as an inhibitor of TGF-beta signaling.

The expression of mouse HtrA1 is developmentally regulated and restricted in embryo tissues which depend largely on TGF-beta signaling for their differentiation. We examined whether mouse HtrA3, another HtrA family member very close to HtrA1, shows similar expression patterns. HtrA3 and -1 were expressed mostly in the same embryonic organs but exhibited complementary patterns in various tissues; the lens epithelial cells in day 12.5 embryo expressed HtrA3 whereas the ciliary body and pigment retina expressed HtrA1. In the vertebrae of day 14.5 embryo, HtrA3 was expressed in the tail region, but HtrA1 was predominantly expressed in the thoracic and lumbar regions. Similar to HtrA1, HtrA3 bound to various TGF-beta proteins and inhibited the signaling of BMP-4, -2 and TGF-beta 1. HtrA3 did not inhibit signaling originated from a constitutively active BMP receptor, indicating that the inhibition occurred upstream of the cell surface receptor. HtrA3 also showed proteolytic activities indistinguishable from those of HtrA1 toward beta-casein and some extracellular matrix (ECM) proteoglycans. The protease activity was absolutely required for the TGF-beta signal inhibition activity. All these data suggest that HtrA3 and -1 have the overlapping biological activities but can function in complementary fashion in certain types of tissues.

HtrA1 serine protease inhibits signaling mediated by Tgfbeta family proteins.

HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, we examined whether HtrA1 functions as an antagonist of Tgfbeta family proteins. During embryo development, mouse HtrA1 was expressed in specific areas where signaling by Tgfbeta family proteins plays important regulatory roles. The GST-pulldown assay showed that HtrA1 binds to a broad range of Tgfbeta family proteins, including Bmp4, Gdf5, Tgfbetas and activin. HtrA1 inhibited signaling by Bmp4, Bmp2, and Tgfbeta1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicated that the binding activity of HtrA1 required the protease domain and a small linker region preceding it, and that inhibition of Tgfbeta signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfbeta family members.