Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
基本信息
- 批准号:6421638
- 负责人:
- 金额:$ 32.31万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2002
- 资助国家:美国
- 起止时间:2002-03-15 至 2006-02-28
- 项目状态:已结题
- 来源:
- 关键词:BCL2 gene /protein antisense nucleic acid bladder neoplasm enzyme activity gene expression gene targeting genetically modified animals method development microarray technology neoplasm /cancer genetics oligonucleotides prostate neoplasms recombinant DNA ribonuclease P thiophosphate tissue /cell culture
项目摘要
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.
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。
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):由于bcl-2基因敲除的前列腺癌细胞和膀胱癌细胞(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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Cy A STEIN', 18)}}的其他基金
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6703068 - 财政年份:2002
- 资助金额:
$ 32.31万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6620770 - 财政年份:2002
- 资助金额:
$ 32.31万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6868959 - 财政年份:2002
- 资助金额:
$ 32.31万 - 项目类别:
Orthogonal Strategies for Specific Knockout Production
特定淘汰赛生产的正交策略
- 批准号:
6823670 - 财政年份:2002
- 资助金额:
$ 32.31万 - 项目类别:
MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES
基因靶向寡核苷酸的分子研究
- 批准号:
2740016 - 财政年份:1999
- 资助金额:
$ 32.31万 - 项目类别:
MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES
基因靶向寡核苷酸的分子研究
- 批准号:
6151246 - 财政年份:1999
- 资助金额:
$ 32.31万 - 项目类别:
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