Orthogonal Strategies for Specific Knockout Production

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DESCRIPTION (provided by applicant): In the "post-genomic era," the need for high through-put specific target validation strategies is immense. The antisense biotechnology may at least partially satisfy this need, but antisense approaches are almost entirely dependent on the phosphorothioate backbone modification. This drastically decreases the specificity of the resultant knockout because of the inherent non-specificity of the phosphorothioates and the reliance on the mRNA-cleaving activity of RNase H. To overcome these problems, we have developed the external guide sequence (EGS) technology, which should produce highly specific knockouts, and requires only a single, specific control sequence. Therefore, we propose: Specific Aim 1: To chemically optimize EGS Activity (Miller lab): To reduce EGS synthesis time, cost, and effort, the stem/loop element common to each EGS and the control reverse loop EGS will be synthesized on a solid support. The hybridizing arms will be subsequently added. Specific Aim 2: Technology Validation: The general applicability of the EGS methodology will be determined (Stein lab): EGSs will extrapolated from the C-raf and A-raf kinase, Ha-ras, and bcl-xL antisense phosphorothioate oligodeoxynucleotide sequences. These four sequences were chosen because they were isolated from a pool of at least 20 candidate molecules, and are hence most likely specific. Experiments will be performed in prostate and bladder carcinoma cells. Specific Aim 3: The specific applicability of the EGS methodology will be determined: We wish to determine why many prostate and bladder carcinoma cells co-express bcl-xL and bcl-2, both strongly anti-apoptotic proteins. Therefore we will evaluate the phenotype of bcl-xL and bcl-2 knockout prostate and bladder carcinoma cells (Stein lab): Because of the non-specificity of the bcl-2 antisense oligonucleotide known as G3139, we must isolate new, specific antisense bcl-2 oligonucleotides by the "mRNA" walking technique, and extrapolate these sequences into a bcl-2 EGS. Biochemical evaluation of apoptosis in the presence or absence of cytotoxic agents in knock-out cells will be accomplished by; evaluation of disruption of inner mitochondrial transmembrane potential (delta-psi-m) and increase in reactive oxygen species; evaluation of cytochrome c levels; and evaluation of pro-caspase- and caspase-3 levels, and ICAD/DFF45 and PARP degradation. The validation of the EGS technology to produce specific knockouts will be an important general contribution to the area of target validation. Finally, in Specific Aim 4, we will evaluate the molecular consequences of the down regulation of bcl-2 and bcl-xL by employing the 12,000 gene-containing Affymetrix oligonucleotide microarray technology.