Functional analysis of secreted and transmembrane proteins critical to mouse development.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.

An LDL-receptor-related protein mediates Wnt signalling in mice.

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.

Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice.

Gene trapping in murine embryonic stem cells is a proven method for the simultaneous identification and mutation of genes in the mouse. Gene trap vectors are designed to detect insertions within genes through the production of a fusion mRNA transcript, making the identification of the endogenous gene possible by 5' rapid amplification of cDNA ends (RACE). Although the amplification of specific cDNAs can be achieved rapidly, cloning and screening of informative-sized cDNAs has proven to be time consuming. To eliminate the need for cloning, we have developed a method for solid-phase sequencing of 5' RACE products. More than 150 independent gene trap cell lines were analyzed, and sequence information was obtained for every line successfully amplified by RACE. With the vector used in this study, 40% of the cell lines were found to contain properly spliced gene trap events. The remaining lines were either spliced inefficiently or contained deletions of the vector. These results highlight the advantage of sequencing gene trap integrations before further characterization. This work now paves the way for large-scale gene trap screens in mice and should greatly accelerate the functional analysis of the mammalian genome.

Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins.

The survival-promoting activity of the Bcl-2 family of proteins appears to be modulated by interactions between various cellular proteins. We have identified a novel cellular protein, Bik, that interacts with the cellular survival-promoting proteins, Bcl-2 and Bcl-xL, as well as the viral survival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirus E1B-19 kDa. In transient transfection assays, Bik promotes cell death in a manner similar to the death-promoting members of the Bcl-2 family, Bax and Bak. This death-promoting activity of Bik can be suppressed by coexpression of Bcl-2, Bcl-XL, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik may be a common target for both cellular and viral anti-apoptotic proteins. While Bik does not show overt homology to the BH1 and BH2 conserved domains characteristic of the Bcl-2 family, it does share a 9 amino acid domain (BH3) with Bax and Bak which may be a critical determinant for the death-promoting activity of these proteins.