RESUMO
I report a method for single nucleotide polymorphism (SNP) detection and linkage mapping in Caenorhabditis elegans using automated oligonucleotide design and fluorescence-based quantitative PCR detection. Nine hundred twenty-three oligonucleotide pairs were designed to produce small products of <150 bp for efficient amplification in a PCR, with one oligonucleotide of each pair overlapping a SNP site at the 3'-most nucleotide. A subset of the pairs were tested, and efficient allelic discrimination was obtained for SNPs between N2, the canonical laboratory strain, and CB4856, a strain isolated from Hawaii commonly used for mapping studies. Linkage mapping is demonstrated using the unc-119 locus of C. elegans. This quantitative PCR method provides an inexpensive, uniform, and automatable detection alternative for genetic mapping strategies in C. elegans or other organisms.
Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , AnimaisRESUMO
We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.