The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) α. We compared the effects of vanadate to PFTα and PFTµ, an inhibitor of transcriptionindependent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcriptionindependent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. Cancer Res; 70(1); 257-65. ©2010 AACR.

Background: Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single-or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. Results: We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single-or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans. Conclusion: Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.

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