Identification of beta-cell-inducing small RNAs by random shRNA selection

通过随机 shRNA 选择鉴定诱导 β 细胞的小 RNA

• 批准号: 7873599
• 负责人: ROBERT B WILSON
• 金额: $ 19.97万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-16 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873599
• 关键词: Address; Back; Beta Cell; Biological; Biological Assay; Cell Differentiation process; Cell Line; Cells; DNA; Development; Diabetes Mellitus; Endocrine; Gene Expression; Gene Targeting; Genes; Genome; Genomics; Green Fluorescent Proteins; Insulin; Libraries; Methods; MicroRNAs; Microarray Analysis; Modeling; Molecular Profiling; Mus; Mutagenesis; Nucleotides; Pancreas; Parents; Protocols documentation; Public Health; RNA; RNA Interference; RNA Sequences; Randomized; Recovery; Reporter; Resistance; Retroviridae; Risk; Screening procedure; Seeds; Small RNA; Sorting - Cell Movement; Stem cells; Technology; Testing; Therapeutic; Transfection; Viral Vector; antibiotic G 418; base; design; embryonic stem cell; improved; new technology; novel therapeutics; protein expression; public health relevance; tissue culture; tool; tumor; vector

DESCRIPTION (provided by applicant): The finding that insulin-secreting, endocrine beta cells can be derived from exocrine cells of the mouse pancreas by expressing Ngn3, Pdx1, and MafA could revolutionize diabetes therapeutics. The primary problem - which this proposal seeks to address - is that the viral vectors used to deliver the genes integrate permanently into the genome, which could interfere with the function of derived cells, or cause tumors. In the induced-stem-cell field, several methods have been tested to obviate the need for vector integration, or the introduction of genes. However, none of these methods is entirely satisfactory. We recently developed a novel technology that has the potential to address the problems described above. This technology is an shRNA-expressing library that is completely random at the nucleotide level. Herein, we propose to use this library to identify shRNA sequences that can reprogram, or increase the efficiency of reprogramming of, endocrine beta cells from exocrine cells of the pancreas. Modulation of gene expression by small RNAs can be accomplished by expression of shRNAs from DNA vectors, or by adding pre-synthesized siRNAs to cells exogenously. The advantage of exogenous siRNAs is that their effects are easy to sustain by repeated addition, and are easily reversible - because there is no vector integration, one can simply stop adding them and allow them to degrade. Thus, effective sequences identified from our random shRNA- encoding library, and optimized by random mutagenesis and re-screening, could be used as pre-synthesized siRNAs, thereby obviating the risks associated with vector integration. In addition, siRNAs are easy to synthesize, and easy to introduce into cells using well-established transfection protocols. PUBLIC HEALTH RELEVANCE: This proposal describes an approach to develop novel therapeutics and biologic tools using an shRNA- expressing library that is completely random at the nucleotide level. This approach has implications for the development of diabetes therapeutics and stem-cell-based therapeutics, and is highly relevant to public health.

描述(申请人提供)：通过表达Ngn3、Pdx1和MafA，可以从小鼠胰腺外分泌细胞获得分泌胰岛素的内分泌β细胞，这一发现可能会使糖尿病的治疗方法发生革命性变化。这项提案试图解决的主要问题是，用于传递基因的病毒载体永久整合到基因组中，这可能会干扰衍生细胞的功能，或导致肿瘤。在诱导干细胞领域，已经测试了几种方法来消除载体整合或基因导入的需要。然而，这些方法都不是完全令人满意的。我们最近开发了一种新技术，有可能解决上述问题。这项技术是一个在核苷酸水平上完全随机的shRNA表达文库。在这里，我们建议使用这个文库来鉴定能够对胰腺外分泌细胞中的内分泌β细胞进行重新编程或提高重新编程效率的shRNA序列。小RNA对基因表达的调控可以通过表达DNA载体上的shRNAs来实现，也可以通过外源添加预先合成的siRNAs到细胞中来实现。外源siRNAs的优势是，它们的作用很容易通过重复添加来维持，并且很容易逆转-因为没有载体整合，人们可以简单地停止添加它们，并允许它们降解。因此，从我们的随机shRNA编码库中鉴定出的有效序列，并通过随机突变和重新筛选进行优化，可以用作预先合成的siRNA，从而避免了与载体整合相关的风险。此外，siRNAs很容易合成，并且很容易通过成熟的转染方案导入细胞。 与公共卫生相关：这项建议描述了一种利用shRNA表达文库开发新疗法和生物工具的方法，该文库在核苷酸水平上是完全随机的。这种方法对糖尿病疗法和基于干细胞的疗法的发展具有影响，并且与公共卫生高度相关。