Orthogonal Strategies for Specific Knockout Production

描述(申请人提供)：在“后基因组时代”，需要 高吞吐量的特定目标验证策略是巨大的。这个 反义生物技术至少可以部分满足这一需求，但反义 这些方法几乎完全依赖于硫代磷酸的骨架。 修改。这极大地降低了结果的特异性。 由于硫代磷酸盐的固有非特异性而导致的基因敲除 依赖核糖核酸酶H的mRNA裂解活性来克服这些 针对这些问题，我们开发了外部引导序列(EGS)技术，该技术 应该产生高度特定的敲除，并且只需要一个单一的、特定的 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和控制反向环路EGS共有的杆/环元素将是 在固体载体上合成。杂交的手臂随后将被 添加了。具体目标2：技术验证： 将确定EGS方法(Stein实验室)：EGSS将从 C-RAF和A-RAF激酶、Ha-ras和bcl-xl反义硫代磷酸 寡核苷酸序列。之所以选择这四个序列，是因为它们 是从至少20个候选分子池中分离出来的，因此 很可能是具体的。实验将在前列腺和膀胱进行 癌细胞。具体目标3：环境影响评估的具体适用性 方法将被确定：我们希望确定为什么许多前列腺癌和 膀胱癌细胞共表达bclxl和bcl2，均为强阳性 抗细胞凋亡蛋白。因此，我们将评估bcl-xl的表型和 BCL-2基因敲除的前列腺和膀胱癌细胞(Stein Lab)：由于 被称为G3139的bcl2反义寡核苷酸的非特异性，我们必须 用“信使核糖核酸”步行法分离新的反义bcl2寡核苷酸 技术，并将这些序列外推到bCL-2 EGS中。生化 细胞毒药物存在或不存在时细胞凋亡的评价 敲除单元将通过以下方式完成；内部破坏的评估 线粒体跨膜电位与反应性增加 氧物种.细胞色素c水平的评估 Caspase-1和caspase-3原水平，ICAD/DFF45和PARP降解。这个 验证EGS技术以生产特定的基因敲除将是 对目标验证领域的重要总体贡献。最后，在 具体目标4，我们将评估倒下的分子后果 利用含12,000个基因对bcl2和bclxl的调控 Affymetrix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.