Orthogonal Strategies for Specific Knockout Production

描述（由申请人提供）：在“后基因组时代”， 高通量特定目标验证策略是巨大的。的 反义生物技术可以至少部分地满足这种需要，但是反义核酸技术可以在一定程度上满足这种需要。 这些方法几乎完全依赖于硫代磷酸酯骨架 改性这大大降低了所得产物的特异性。 由于硫代磷酸酯固有的非特异性， 依赖RNase H的mRNA切割活性。克服这些 问题，我们已经开发了外部指导序列（EGS）技术， 应该产生高度特异性的敲除，并且只需要一个单一的、特异的 控制序列因此，我们提出：具体目标1：化学优化 EGS活动（米勒实验室）：为了减少EGS合成时间、成本和工作量， 每个EGS和控制反向回路EGS共用的茎/回路元件将 在固体载体上合成。杂交臂随后将被 补充说具体目标2：技术验证： 将确定EGS方法（Stein实验室）：EGS将从 C-raf和A-raf激酶、Ha-ras和bcl-xL反义硫代磷酸酯 寡脱氧核苷酸序列。选择这四个序列是因为它们 从至少20个候选分子的库中分离，因此 很可能是具体的。实验将在前列腺和膀胱中进行 癌细胞具体目标3：环境商品和服务的具体适用性 方法将被确定：我们希望确定为什么许多前列腺和 膀胱癌细胞共表达bcl-xL和bcl-2，两者均强表达， 抗凋亡蛋白。因此，我们将评估bcl-xL的表型， bcl-2基因敲除的前列腺癌和膀胱癌细胞（Stein实验室）： bcl-2反义寡核苷酸G3139的非特异性，我们必须 通过“mRNA”步移分离新的、特异性的反义bcl-2寡核苷酸 技术，并将这些序列外推到bcl-2 EGS中。生化 在存在或不存在细胞毒性剂的情况下评价细胞凋亡， 敲除细胞将通过以下方式完成：评价内 线粒体跨膜电位（delta-psi-m）和反应性 氧物种;评价细胞色素c水平;和评价 胱天蛋白酶原和胱天蛋白酶-3水平以及ICAD/DFF 45和PARP降解。的 EGS技术的验证，以产生特定的敲除将是 对目标验证领域的重要贡献。最后在 具体目标4，我们将评估分子的后果， 通过使用含有12，000个基因的 亲和寡核苷酸微阵列技术。

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.