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

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) f lanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green f luorescent protein transgene that had been designed to contain a single base deletion. Recovery of f luorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.Genomics is currently a central component of plant biology research. The gene sequence of Arabidopsis thaliana will soon be available, and many other species are under study. Identification of new genes is occurring at a much faster pace than is the determination of their function. One missing technology in plant biology is the ability to selectively and reliably create site-specific ''gene knockouts'' or homologous recombination of genes of interest or of unknown function. The phenomena of gene silencing (e.g., antisense and cosuppression) provides a method for understanding gene function through the creation of transgenic crops expressing a gene sequence that silences the endogenous gene. However, this approach is not suitable for functional genomic studies of individual members of multigene families or genes with similar sequence, and is sometimes problematic in other technical aspects (1).A technology currently being explored in prokaryotic and eukaryotic systems uses self-complementary chimeric oligonucleotides (COs) comprised of DNA and 2Ј-O-methyl RNA to target and mutate genes in vivo. These COs are designed to have one or more bases that do not pair with the endogenous gene sequence. This approach was successfully used to modify endogenous genes of mammalian cells (2-5) in a site-specific and genetically inheritable manner. Recently, Alexeev and Yoon (5) have demonstrated that permanent in vivo conversions result in phenotypic changes in mouse melanocytes. It has been hypothesized that the mechan...

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.Chimeric RNA/DNA oligonucleotides (chimeras) have been used to direct single base changes in episomal and chromosomal targets in mammalian cells Yoon et al., 1996; Alexeev and Yoon, 1998;Kren et al., 1998;Lai and Lien, 1999). These molecules have also been effective in mediating similar reactions in plant cells (Beetham et al., 1999;Zhu et al., 1999). Independently, these two groups demonstrated that mutations can be corrected in tobacco (Nicotiana tabacum) and maize (Zea mays) cells. Using a marker gene system, Zhu et al. (1999) corrected a mutation in vivo and showed that the targeted base was in fact altered as directed by the chimeric oligonucleotide. Although Beetham et al. (1999) were able to recover herbicide-resistant plant cells due to the action of the chimera, the targeted base in tobacco cells was not changed. Instead, the nucleotide located at the 5Ј side of the target base was mutated. Because such a change would also produce an amino acid alteration that would confer herbicide resistance, the plant cells were recoverable under the appropriate selection. These results were the first to demonstrate degeneracy in targeted gene repair because experiments in mammalian cells produced only precise nucleotide alterations.The process by which these nucleotide conversions are made is still undefined, but recent evidence suggests that mismatch repair plays a critical role in mammalian cells. Using c...

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