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.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34 ؉ CD38 ؊ CD90 ؉ Lin ؊ ) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.JAK ͉ signaling ͉ progenitors ͉ cell fate ͉ mutant allele

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Aquaporin-0 (AQP0), a water transport channel protein, is the major intrinsic protein (MIP) of lens fiber cell plasma membranes. Mice deficient in the gene for AQP0 (Aqp0, Mip) were generated from a library of gene trap embryo stem cells. Sequence analysis showed that the gene trap vector had inserted into the first exon of Aqp0, causing a null mutation as verified by RNA blotting and immunochemistry. At 3 wk of age (postnatal day 21), lenses from null mice (Aqp0(-/-)) contained polymorphic opacities, whereas lenses from heterozygous mice (Aqp0(+/-)) were transparent and did not develop frank opacities until approximately 24 wk of age. Osmotic water permeability values for Aqp0(+/-) and Aqp0(-/-) lenses were reduced to approximately 46% and approximately 20% of wild-type values, respectively, and the focusing power of Aqp0(+/-) lenses was significantly lower than that of wild type. These findings show that heterozygous loss of AQP0 is sufficient to trigger cataractogenesis in mice and suggest that this MIP is required for optimal focusing of the crystalline lens.

Tumor-associated DNA has been detected in plasma of colorectal cancer (CRC) patients using various techniques but with limited gene or mutation coverage. We report a highly sensitive scanning methodology for mutational assessment of the APC and TP53 genes, which typically pose an analytical challenge because of their significant genotypic heterogeneity as well as specific mutational scoring assays for K-RAS and BRAF. Plasma DNA isolated from 20 CRC patients were scanned for mutations in these targets without knowledge of the molecular or pathological analyses of the matched primary tumors. We chose mutation scanning technology and these molecular targets to provide a comprehensive screen for somatic mutations known to be associated with sporadic CRC. Mutations were identified with a novel denaturing high-performance liquid chromatography (DHPLC) platform that uses post-separation fluorescence technology to enable the detection of variants that represent <0.1% of the total analyzed DNA. Mutant allele specific amplification (MASA) followed by detection with the same platform was used to identify low-level target mutations (mutation scoring) in K-RAS codons 12, 13, and 61, and BRAF codon 599. Using this combined scanning and scoring approach, we were able to identify at least one mutational event in 20/20 (100%) CRC patients. The thoroughness of a mutation scanning and scoring panel may have important implications for CRC screening and disease monitoring during and following therapy.