Inhibition of Male Germ Cell Proteins by RNAi

RNAi 对雄性生殖细胞蛋白的抑制

• 批准号: 6890956
• 负责人: STUART B MOSS
• 金额: $ 7.93万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890956
• 关键词: RNA interference; apoptosis; double stranded RNA; gene expression; gene targeting; genetically modified animals; germ cells; immunofluorescence technique; laboratory mouse; messenger RNA; polymerase chain reaction; protein structure function; small interfering RNA; sperm; spermatogenesis; terminal nick end labeling

DESCRIPTION (provided by applicant): Determining the function of proteins in male germ cells and/or mature sperm is difficult. While gene disruption by homologous recombination is specific and completely eliminates a particular protein, it is a labor-intensive process. Given that a mutant phenotype may not be observed due to genetic redundancy, it is also a risky approach. The introduction of a 500-600 nt double-stranded RNA (dsRNA) that has sequence homology to a particular gene suppresses its expression. These dsRNAs are processed into short (19-25 nt) interfering RNAs (siRNAs) who prompt the specific degradation of homologous cellular mRNA (RNA interference). While the introduction of dsRNAs longer than 30 nt into mammalian somatic cells can elicit a non-specific apoptotic response, short siRNAs causes gene-specific silencing without inducing apoptosis. The effectiveness of siRNA and dsRNA to eliminate/reduce a gene product in mouse male germ cells will be examined using AKAP4 as a "model" protein target. AKAP4, a protein in the sperm fibrous sheath, has several advantages: 1) reagents including antibodies against the protein are available, 2) the gene has been disrupted by homologous recombination so it can be determined if AKAP4 reduction by siRNA/dsRNA "phenocopies" the null, and 3) the generation of a mutant phenotype by siRNA/dsRNA of an abundant protein such as AKAP4 suggests that the function of other spermatogenic proteins can be studied similarly. AKAP4 will be reduced/eliminated in spermatids and sperm by two approaches. In the first, siRNA corresponding to a region of AKAP4 will be transcribed from the RNA polymerase III promoter, U6. One possible shortcoming of this approach is that the ubiquitously-expressed U6 promoter can not be used to examine proteins that are expressed in both the testis and other tissues (this is not a problem for the spermatid-specific AKAP4). To circumvent this limitation, dsRNA corresponding to AKAP4 also will be expressed from the spermatid-specific protamine promoter to examine whether AKAP4 can be reduced/eliminated without eliciting a non-specific apoptotic response. This siRNA/dsRNA method would provide an extremely powerful approach for examining the function of proteins expressed during spermatogenesis. It is a relatively simple technique, requiring the establishment of a transgenic line. Furthermore, if the RNAi/dsRNA approach "knocks-down" but does not "knock-out" a particular protein, hypomorph phenotypes may be produced that will provide additional information about gene function, e.g., the threshold levels at which proteins function properly.

描述（由申请人提供）：确定男性生殖细胞和/或成熟精子中蛋白质的功能很困难。虽然同源重组的基因破坏是特异性的并且完全消除了特定的蛋白质，但这是一个劳动密集型的过程。鉴于由于遗传冗余而可能无法观察到突变表型，这也是一种危险的方法。引入与特定基因具有序列同源性的 500-600 nt 双链 RNA (dsRNA) 可抑制其表达。这些 dsRNA 被加工成短的 (19-25 nt) 干扰 RNA (siRNA)，促进同源细胞 mRNA 的特异性降解（RNA 干扰）。虽然将长度超过 30 nt 的 dsRNA 引入哺乳动物体细胞可以引发非特异性细胞凋亡反应，但短 siRNA 会导致基因特异性沉默而不诱导细胞凋亡。将使用 AKAP4 作为“模型”蛋白质靶点来检查 siRNA 和 dsRNA 消除/减少小鼠雄性生殖细胞中基因产物的有效性。 AKAP4 是精子纤维鞘中的一种蛋白质，具有几个优点：1) 可以使用包括针对该蛋白质的抗体的试剂，2) 该基因已通过同源重组被破坏，因此可以确定 siRNA/dsRNA 减少 AKAP4 是否“表型复制”无效，以及 3) 通过 siRNA/dsRNA 产生丰富蛋白质的突变表型，例如 AKAP4 表明其他生精蛋白的功能也可以进行类似的研究。通过两种方法可以减少/消除精细胞和精子中的 AKAP4。首先，对应于 AKAP4 区域的 siRNA 将从 RNA 聚合酶 III 启动子 U6 转录。这种方法的一个可能的缺点是普遍表达的 U6 启动子不能用于检查在睾丸和其他组织中表达的蛋白质（这对于精子细胞特异性 AKAP4 来说不是问题）。为了规避这一限制，对应于 AKAP4 的 dsRNA 也将从精子特异性鱼精蛋白启动子中表达，以检查 AKAP4 是否可以被减少/消除而不引发非特异性凋亡反应。这种 siRNA/dsRNA 方法将为检查精子发生过程中表达的蛋白质的功能提供极其强大的方法。这是一种相对简单的技术，需要建立转基因系。此外，如果RNAi/dsRNA接近“敲低”但不“敲除”特定蛋白质，则可能产生亚型体表型，这将提供有关基因功能的额外信息，例如蛋白质正常发挥作用的阈值水平。