Inhibition of Male Germ Cell Proteins by RNAi

描述（由申请人提供）：确定雄性生殖细胞和/或成熟精子中蛋白质的功能是困难的。虽然通过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过程。鉴于突变表型可能由于遗传冗余而无法观察到，因此这也是一种有风险的方法。与特定基因具有序列同源性的500-600 nt双链RNA（dsRNA）的引入抑制其表达。这些dsRNA被加工成短（19-25 nt）干扰RNA（siRNA），其促进同源细胞mRNA的特异性降解（RNA干扰）。虽然将长于30 nt的dsRNA引入哺乳动物体细胞中可以引发非特异性凋亡应答，但短siRNA引起基因特异性沉默而不诱导凋亡。将使用AKAP 4作为“模型”蛋白靶标来检查siRNA和dsRNA消除/减少小鼠雄性生殖细胞中的基因产物的有效性。AKAP 4是精子纤维鞘中的一种蛋白质，具有以下几个优点：1）包括针对蛋白质的抗体的试剂是可获得的，2）基因已经通过同源重组被破坏，因此可以确定是否通过siRNA/dsRNA的AKAP 4还原“表型复制”无效，和3）通过siRNA/大量蛋白质如AKAP 4的dsRNA表明其他生精蛋白的功能可以类似地进行研究。AKAP 4将通过两种方法在精子细胞和精子中减少/消除。在第一种情况下，对应于AKAP 4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启动子不能用于检测在睾丸和其他组织中表达的蛋白质（这对于精子特异性AKAP 4来说不是问题）。为了规避这种限制，也将从精子细胞特异性鱼精蛋白启动子表达对应于AKAP 4的dsRNA，以检查AKAP 4是否可以被减少/消除而不引发非特异性凋亡应答。这种siRNA/dsRNA方法将提供一个非常强大的方法来检查在精子发生过程中表达的蛋白质的功能。这是一种相对简单的技术，需要建立转基因品系。此外，如果RNAi/dsRNA方法“敲低”但不“敲除”特定蛋白质，则可能产生亚型表型，其将提供关于基因功能的额外信息，例如，蛋白质正常发挥功能的阈值水平。

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.