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1. 发明授权

US08895525B2 Preventing hyaluronan-mediated tumorigenetic mechanisms using intronic RNAs 有权
标题翻译：使用内含子RNA预防透明质酸介导的肿瘤发生机制
公开(公告)号：US08895525B2
公开(公告)日：2014-11-25
申请号：US12740334
申请日：2008-10-29
申请人： Shao-Yao Ying , Shi-Lung Lin
发明人： Shao-Yao Ying , Shi-Lung Lin
IPC分类号： A61K48/00 , C07H21/02 , C07H21/04
CPC分类号： C12N15/1135 , C12N2310/141 , C12Q1/6886 , C12Q2600/112 , C12Q2600/178
摘要： Patterns of microRNA (miRNA) expression are correlated to the degrees of tumor cell differentiation in human prostate cancer. MiRNAs can complementarily bind to either oncogenes or tumor suppressor genes, resulting in targeted gene silencing and thus changes of cellular tumorigenecity. Using miRNA microarray analysis, 8 down-regulated and 3 up-regulated known miRNAs in androgen-independent human prostate cancer cell lines, such as LNCaP C4-2B and PC3, compared to those androgen-dependent cell lines, such as LNCaP and PC3-AR9 were consistently detected. Fluorescent in-situ hybridization assays in human prostate cancer tissue arrays containing sixty patients at different stages also showed the same miRNA expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive non-cancerous prostate epithelium. In-vitro tumorigenecity assays using one of the identified miRNAs, mir-146a, were performed to provide validation of its function in prostate cancer. Gain-of-function transfection of mir-146a markedly suppressed its targeted ROCK1 gene expression in androgen-independent PC3 cells, consequently resulting in reduced cancer cell proliferation, invasion and metastasis to human bone marrow endothelial cell monolayers. Since ROCK1 is the key kinase for activating hyaluronan-mediated HRPC transformation in vivo and in PC3 cells, mir-146a should function as a tumor-suppressor gene in modulating the ROCK1-associated tumorigenecity.
摘要翻译： microRNA（miRNA）表达的模式与人类前列腺癌的肿瘤细胞分化程度相关。 MiRNA可以互补结合癌基因或肿瘤抑制基因，导致靶向基因沉默，从而导致细胞致瘤性的变化。与雄激素依赖性细胞系（如LNCaP和PC3）相比，使用miRNA微阵列分析，8个下调和3个上调已知的与雄激素非依赖性前列腺癌细胞系（如LNCaP C4-2B和PC3）的miRNA相比，一直检测到AR9。与雄激素敏感的非癌前列腺上皮相比，含有60位不同阶段患者的人类前列腺癌组织阵列中的荧光原位杂交测定也显示与激素难治性前列腺癌（HRPC）相同的miRNA表达模式。使用鉴定的miRNA之一的mir-146a进行体外肿瘤发生测定，以提供其在前列腺癌中的功能的验证。 mir-146a功能的功能转染显着抑制雄激素依赖性PC3细胞中靶向的ROCK1基因表达，从而导致癌细胞增殖，侵袭和转移到人类骨髓内皮细胞单层。由于ROCK1是在体内和PC3细胞中激活透明质酸介导的HRPC转化的关键激酶，因此mir-146a应该作为调节ROCK1相关致瘤性的肿瘤抑制基因起作用。

2. 发明授权

US08372969B2 RNA interference methods using DNA-RNA duplex constructs 有权
标题翻译：使用DNA-RNA双链体构建的RNA干扰方法
公开(公告)号：US08372969B2
公开(公告)日：2013-02-12
申请号：US12911654
申请日：2010-10-25
申请人： Shao-Yao Ying , Shi-Lung Lin
发明人： Shao-Yao Ying , Shi-Lung Lin
IPC分类号： C07H21/04
CPC分类号： C12N15/1096
摘要： The present invention provides novel compositions and methods for suppressing the function or activity of a targeted gene through a novel intracellular piRNA-mediated RNAi mechanism, using RNA-DNA duplex constructs. The invention further provides novel methods and compositions for generating or producing RNA-DNA duplex agents, whose quantity is high enough to be used for the invention's gene silencing transfection and possibly in therapeutics applications. This improved RNA-polymerase chain reaction (RNA-PCR) method utilizes thermocycling steps of promoter-linked DNA or RNA template synthesis, in vitro transcription and then reverse transcription to bring up the amount of RNA-DNA duplexes up to two thousand folds within one round of the above procedure for using in D-RNAi-directed gene silencing.
摘要翻译：本发明提供了使用RNA-DNA双链构建体通过新的细胞内piRNA介导的RNAi机制抑制靶基因的功能或活性的新型组合物和方法。本发明还提供用于产生或产生RNA-DNA双链体的新方法和组合物，其量足够高以用于本发明的基因沉默转染并且可能在治疗应用中。这种改进的RNA聚合酶链反应（RNA-PCR）方法利用启动子连接的DNA或RNA模板合成的热循环步骤，体外转录，然后逆转录，将RNA-DNA双链体的量在一个以上用于D-RNAi定向基因沉默的方法。

3. 发明授权

US5928872A Subtractive hybridization with covalently binding homology 失效
标题翻译：具有共价结合同源性的消减杂交
公开(公告)号：US5928872A
公开(公告)日：1999-07-27
申请号：US927859
申请日：1997-09-11
申请人： Shi-Lung Lin , Shao-Yao Ying
发明人： Shi-Lung Lin , Shao-Yao Ying
IPC分类号： C12N15/10 , C12Q1/68 , C12P19/34
CPC分类号： C12N15/1034 , C12Q1/6809
摘要： Excess amount of modified subtracter DNA from control cells is generated by carboxylating the base structures of its certain nucleotides with chemical agents in order to introduce covalent affinity between the modified subtracter and a non-modified tester DNA. Hybridization of the control subtracter and the experimental tester DNA is performed with a heat-melting and then cool-reassociation technique. While the desired different (heterologous) sequences remain in the form of hydrogen-binding, common (homologous) sequences of the hybridized DNA are covalently bonded to each other. Since the covalent bonding of the common sequences can not be broken during a polymerase chain reaction, resulting in no amplification of the common sequences but great amplification of the desired different sequences. The desired DNA sequences present after such covalent homologue subtraction and selective amplification represent those DNA sequences which only exist in the tester but not in the subtracter DNA library.
摘要翻译：通过用化学试剂羧化其某些核苷酸的碱基结构来产生来自对照细胞的过量的修饰的减毒DNA，以便在修饰的减毒剂和未修饰的测试DNA之间引入共价亲和力。控制减法器和实验检测器DNA的杂交用热熔化然后冷再结合技术进行。尽管期望的不同（异源）序列以氢结合的形式保留，杂交DNA的共同（同源）序列彼此共价键合。由于在聚合酶链反应期间共同序列的共价键不能被破坏，所以不能扩增常见序列，而是扩增所需的不同序列。在这种共价同系物减法和选择性扩增之后存在的所需DNA序列代表仅存在于测试者中但不在减毒菌DNA文库中的那些DNA序列。

4. 发明授权

US5871927A Nucleotide analog-containing hybrid subtraction with sequentially enzymatic digestion 失效
标题翻译：含有序列酶消化的含核苷酸类似物的杂交减法
公开(公告)号：US5871927A
公开(公告)日：1999-02-16
申请号：US854400
申请日：1997-05-12
申请人： Shi-Lung Lin , Shao-Yao Ying
发明人： Shi-Lung Lin , Shao-Yao Ying
IPC分类号： C12N15/10 , C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6809 , C12N15/1034
摘要： The present invention provides a method for fast, simple, and reliable isolation of desired different sequences from two DNA libraries. Excess amount of nucleotide analog-containing DNA subtracter from control cells is generated by incorporating nucleotide analog with a template-dependent extension reaction to introduce susceptible-sites for subsequent enzymatic digestion. Hybridization of the control subtracter and experimental DNA is performed with a heat-melting and then cool-reassociation technique. The hybridized DNAs are subtracted with nucleotide analog-removing enzyme first, resulting in nicking or gapping all nucleotide analog-containing hybrid duplexes which are further digested by single-strand-specific nuclease. Desired DNA sequences from the experimental cells, but not the control ones stay intact throughout the digestion procedure and can be selectively amplified at the end. This technique is designed for the subtractive hybridization of different sequences between two DNA libraries from distinct cell sources and will allow more efficient isolations in experiments on cancer formation, development of gene therapy, and understanding of pathological status and developmental regulation.
摘要翻译：本发明提供了一种用于从两个DNA文库快速，简单和可靠地分离所需不同序列的方法。通过将具有模板依赖性延伸反应的核苷酸类似物引入来自对照细胞的含核苷酸类似物的DNA减法器的多量来引入敏感位点用于随后的酶消化。控制减法器和实验DNA的杂交通过加热熔化然后冷却重新连接技术进行。首先用核苷酸类似物去除酶减去杂交的DNA，导致所有含有核苷酸类似物的杂交双链体的切口或间隙，其由单链特异性核酸酶进一步消化。来自实验细胞的所需DNA序列，但不是对照物在整个消化过程中保持完整，并且可以在末端选择性扩增。该技术设计用于来自不同细胞来源的两个DNA文库之间的不同序列的消减杂交，并且将允许在癌症形成，基因治疗的发展，病理状态和发育调节的理解的实验中更有效的分离。

5. 发明授权

US08609831B2 RNA-mediated gene modulation 失效
标题翻译： RNA介导的基因调控
公开(公告)号：US08609831B2
公开(公告)日：2013-12-17
申请号：US10663875
申请日：2003-09-16
申请人： Shi-Lung Lin , Shao-Yao Ying
发明人： Shi-Lung Lin , Shao-Yao Ying
IPC分类号： C07H21/04
CPC分类号： C12N15/113 , C12N15/63 , C12N2310/14 , C12N2310/30
摘要： An isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene. Also disclosed are a composition comprising a chemokine and an isolated RNA of the invention or a DNA template for the isolated RNA, a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell, and methods of using these compositions for modulating the functions of genes in a cell.
摘要翻译：一种分离的RNA，其包含在细胞中释放的内含子RNA，从而调节靶基因的功能。还公开了包含本发明的趋化因子和分离的RNA或分离的RNA的DNA模板的组合物，包含诱导RNA介导的调节细胞中两种或更多种靶基因的功能的一种或多种试剂的组合物，以及使用这些组合物调节细胞中基因功能的方法。

6. 发明申请

US20100298416A1 PREVENTING HYALURONAN-MEDIATED TUMORIGENETIC MECHANISMS USING INTRONIC RNAS 有权
标题翻译：使用内源性RNA酶预防血浆介导的肿瘤机制
公开(公告)号：US20100298416A1
公开(公告)日：2010-11-25
申请号：US12740334
申请日：2008-10-29
申请人： Shao-Yao Ying , Shi-Lung Lin
发明人： Shao-Yao Ying , Shi-Lung Lin
IPC分类号： C12Q1/68 , A61K31/7105
CPC分类号： C12N15/1135 , C12N2310/141 , C12Q1/6886 , C12Q2600/112 , C12Q2600/178
摘要： Patterns of microRNA (miRNA) expression are correlated to the degrees of tumor cell differentiation in human prostate cancer. MiRNAs can complementarily bind to either oncogenes or tumor suppressor genes, resulting in targeted gene silencing and thus changes of cellular tumorigenecity. Using miRNA microarray analysis, 8 down-regulated and 3 up-regulated known miRNAs in androgen-independent human prostate cancer cell lines, such as LNCaP C4-2B and PC3, compared to those androgen-dependent cell lines, such as LNCaP and PC3-AR9 were consistently detected. Fluorescent in-situ hybridization assays in human prostate cancer tissue arrays containing sixty patients at different stages also showed the same miRNA expression patterns in hormone-refractory prostate carcinomas (HRPC) compared to androgen-sensitive non-cancerous prostate epithelium. In-vitro tumorigenecity assays using one of the identified miRNAs, mir-146a, were performed to provide validation of its function in prostate cancer. Gain-of-function transfection of mir-146a markedly suppressed its targeted ROCK1 gene expression in androgen-independent PC3 cells, consequently resulting in reduced cancer cell proliferation, invasion and metastasis to human bone marrow endothelial cell monolayers. Since ROCK1 is the key kinase for activating hyaluronan-mediated HRPC transformation in vivo and in PC3 cells, mir-146a should function as a tumor-suppressor gene in modulating the ROCK1-associated tumorigenecity.
摘要翻译： microRNA（miRNA）表达的模式与人类前列腺癌的肿瘤细胞分化程度相关。 MiRNA可以互补结合癌基因或肿瘤抑制基因，导致靶向基因沉默，从而导致细胞致瘤性的变化。与雄激素依赖性细胞系（如LNCaP和PC3）相比，使用miRNA微阵列分析，8个下调和3个上调已知的与雄激素非依赖性前列腺癌细胞系（如LNCaP C4-2B和PC3）的miRNA相比，一直检测到AR9。与雄激素敏感的非癌前列腺上皮相比，含有60位不同阶段患者的人类前列腺癌组织阵列中的荧光原位杂交测定也显示与激素难治性前列腺癌（HRPC）相同的miRNA表达模式。使用鉴定的miRNA之一的mir-146a进行体外肿瘤发生测定，以提供其在前列腺癌中的功能的验证。 mir-146a功能的功能转染显着抑制雄激素依赖性PC3细胞中靶向的ROCK1基因表达，从而导致癌细胞增殖，侵袭和转移到人类骨髓内皮细胞单层。由于ROCK1是在体内和PC3细胞中激活透明质酸介导的HRPC转化的关键激酶，因此mir-146a应该作为调节ROCK1相关致瘤性的肿瘤抑制基因。

7. 发明授权

US06197554B1 Method for generating full-length cDNA library from single cells 失效
标题翻译：从单细胞产生全长cDNA文库的方法
公开(公告)号：US06197554B1
公开(公告)日：2001-03-06
申请号：US09197951
申请日：1998-11-20
申请人： Shi-Lung Lin , Cheng-Ming Chuong , Shao-Yao Ying
发明人： Shi-Lung Lin , Cheng-Ming Chuong , Shao-Yao Ying
IPC分类号： C12P1934
CPC分类号： C12N15/1093 , C12N15/1096
摘要： The present invention provides a fast, simple and specific method for generating a complete full-length cDNA library from single cells. The first reverse transcription of intracellular mRNAs with an oligo(dT)n-promoter primer introduces a recognition site for following transcription of newly reverse-transcribed cDNAs. The poly-nucleotide tailing of above cDNAs in addition to aforementioned promoter region further forms binding templates for specific PCR amplification. After repeating the reverse transcription, transcription, reverse transcription and PCR procedure, we can multiply a single copy of mRNA to two billion folds by calculation based upon the comparison between the amount of a synthesized cDNA library and that of theoretically presumed mRNAs within a cell (0.1 pg). In conjunction with a cell fixation and permeabilization step, the complete full-length cDNA library can be directly generated from few single cells without mRNA degradation. The present invention will be very useful in preparing tissue-specific full-length cDNA libraries for future gene chip technology.
摘要翻译：本发明提供了用于从单细胞产生完整全长cDNA文库的快速，简单和具体的方法。用oligo（dT）n启动子引物进行细胞内mRNA的第一次逆转录引入了新的逆转录cDNAs的后续转录的识别位点。除了上述启动子区域之外，上述cDNA的多核苷酸尾料进一步形成用于特异性PCR扩增的结合模板。在重复逆转录，转录，逆转录和PCR过程之后，我们可以基于合成的cDNA文库的量与细胞内理论假定的mRNA的量进行比较，通过计算将单拷贝的mRNA增加到20亿倍（ 0.1pg）。结合细胞固定和透化步骤，完整的全长cDNA文库可以直接从少数单细胞产生而不会降解mRNA。本发明在制备用于将来的基因芯片技术的组织特异性全长cDNA文库中将是非常有用的。

8. 发明授权

US6015676A Method for knocking out gene transcripts by covalently binding of an anti-sense probe 失效
标题翻译：通过共价结合反义探针敲除基因转录物的方法
公开(公告)号：US6015676A
公开(公告)日：2000-01-18
申请号：US127368
申请日：1998-07-31
申请人： Shi-Lung Lin , Shao-Yao Ying
发明人： Shi-Lung Lin , Shao-Yao Ying
IPC分类号： A61K38/00 , A61K48/00 , C12N15/113 , C12N15/63 , C12Q1/68 , C12P19/34
CPC分类号： C12N15/113 , C12N15/63 , A61K38/00 , A61K48/00 , C12N2310/33 , Y10S435/81
摘要： The present invention provides a simple, specific and nontoxic gene knock-out method by formation of covalently bonding between modified probes and targeted sequences. When covalently modified first strand probes are hybridized with a second strand of targeted gene transcripts, certain modified bases of said first strand will interact with natural bases of said second strand to form covalent bonds by which the translation of said second strand is inhibited. Because the hybridization of said two strands generates covalent base-pairing only between their complementary homology region(s), such specificity increases the targeting efficiency of a gene knock-out system. Also, because neither a polymerase extension reaction nor a nuclease digestion can be performed through the covalently bonded region(s) of aforesaid hybrids, the present invention in conjunction with a delivery method can be used to inactivate intracellular functions of targeted nucleotide sequences, to inhibit viral infections in vivo and to increase binding stability of antisense drugs in a gene therapy.
摘要翻译：本发明通过形成经修饰的探针和靶向序列之间的共价键来提供简单，特异和无毒的基因敲除方法。当共价修饰的第一链探针与靶基因转录物的第二链杂交时，所述第一链的某些修饰的碱基将与所述第二链的天然碱基相互作用以形成所述第二链的翻译被抑制的共价键。因为所述两条链的杂交仅在其互补同源区之间产生共价碱基配对，所以这种特异性增加了基因敲除系统的靶向效率。此外，由于不能通过上述杂交体的共价键合区域进行聚合酶延伸反应和核酸酶消化，本发明结合递送方法可以使靶向核苷酸序列的细胞内功能失活，抑制病毒感染在体内并增加反义药物在基因治疗中的结合稳定性。

9. 发明授权

US09567591B2 Generation of human embryonic stem-like cells using intronic RNA 有权
标题翻译：使用内含子RNA产生人胚胎干细胞样细胞
公开(公告)号：US09567591B2
公开(公告)日：2017-02-14
申请号：US12149725
申请日：2008-05-07
申请人： Shi-Lung Lin , Shao-Yao Ying , David Ts Wu
发明人： Shi-Lung Lin , Shao-Yao Ying , David Ts Wu
IPC分类号： C12N15/11 , C07H21/02 , C07H21/04 , C12Q1/68 , C12N5/02 , C12N15/64 , C12N15/63
CPC分类号： C12N15/63
摘要： This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.
摘要翻译：本发明一般涉及使用内含子微RNA样RNA试剂的转基因表达来开发，产生和选择人胚胎干（hES）样多能细胞的方法。更具体地说，本发明涉及一种用于产生非天然存在的内含子及其内含子组分的方法和组合物，其能够在哺乳动物细胞中加工成mir-302样RNA分子，从而诱导对分化 - 相关和命运决定基因，导致将细胞重编程为多能胚胎干（ES）细胞样状态。如此获得的ES样细胞强烈表达hES细胞标志物，例如Oct3 / 4，SSEA-3和SSEA-4，并且可以通过在饲养层上处理某些激素和/或生长因子而被引导到各种组织细胞类型中，体外免疫细胞培养条件，可用于移植和基因治疗。因此，本发明提供了一种简单，有效和安全的基因操作方法，用于不仅将体细胞重编程为ES样多能细胞，而且有助于在无饲养细胞培养条件下维持ES细胞的多能性和更新性质，预防将以前的iPS方法中使用的四个大转录因子基因的繁琐的逆转录病毒插入一个单细胞中。

10. 发明申请

US20110098461A1 Novel RNA Interference Methods Using DNA-RNA Duplex Constructs 有权
标题翻译：使用DNA-RNA复合构建体的新型RNA干扰方法
公开(公告)号：US20110098461A1
公开(公告)日：2011-04-28
申请号：US12911654
申请日：2010-10-25
申请人： Shao-Yao Ying , Shi-Lung Lin
发明人： Shao-Yao Ying , Shi-Lung Lin
IPC分类号： C07H21/02
CPC分类号： C12N15/1096
摘要： The present invention provides novel compositions and methods for suppressing the function or activity of a targeted gene through a novel intracellular piRNA-mediated RNAi mechanism, using RNA-DNA duplex constructs. The invention further provides novel methods and compositions for generating or producing RNA-DNA duplex agents, whose quantity is high enough to be used for the invention's gene silencing transfection and possibly in therapeutics applications. This improved RNA-polymerase chain reaction (RNA-PCR) method utilizes thermocycling steps of promoter-linked DNA or RNA template synthesis, in vitro transcription and then reverse transcription to bring up the amount of RNA-DNA duplexes up to two thousand folds within one round of the above procedure for using in D-RNAi-directed gene silencing.
摘要翻译：本发明提供了使用RNA-DNA双链构建体通过新的细胞内piRNA介导的RNAi机制抑制靶基因的功能或活性的新型组合物和方法。本发明还提供用于产生或产生RNA-DNA双链体的新方法和组合物，其量足够高以用于本发明的基因沉默转染并且可能在治疗应用中。这种改进的RNA聚合酶链反应（RNA-PCR）方法利用启动子连接的DNA或RNA模板合成的热循环步骤，体外转录，然后逆转录，将RNA-DNA双链体的量在一个以上用于D-RNAi定向基因沉默的方法。

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