Inhibition of Male Germ Cell Proteins by RNAi

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是困难的。虽然同源重组的基因破坏是特异性的，完全消除了特定的蛋白质，但这是一个劳动密集型的过程。考虑到由于基因冗余，可能无法观察到突变表型，这也是一种危险的方法。引入与特定基因序列同源的500-600 nt双链RNA （dsRNA）可抑制其表达。这些dsRNAs被加工成短的（19- 25nt）干扰RNA (sirna)， sirna促进同源细胞mRNA的特异性降解（RNA干扰）。将长度超过30 nt的dsRNAs引入哺乳动物体细胞可引起非特异性凋亡反应，而短sirna可引起基因特异性沉默而不诱导细胞凋亡。以AKAP4作为“模型”蛋白靶点，研究siRNA和dsRNA在小鼠雄性生殖细胞中消除/减少基因产物的有效性。AKAP4是精子纤维鞘中的一种蛋白，具有以下几个优点：1)可获得包括针对该蛋白的抗体在内的试剂；2)该基因已被同源重组破坏，因此可以确定siRNA/dsRNA对AKAP4的还原是否“表型”缺失；3)siRNA/dsRNA对AKAP4等丰富蛋白产生突变表型，表明可以类似地研究其他生精蛋白的功能。AKAP4将通过两种途径在精细胞和精子中减少/消除。首先，从RNA聚合酶III启动子U6转录对应AKAP4区域的siRNA。这种方法的一个可能的缺点是，无处不在表达的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（这对于精子特异性AKAP4来说不是问题）。为了规避这一限制，AKAP4对应的dsRNA也将从精细胞特异性鱼精蛋白启动子中表达，以检查AKAP4是否可以在不引起非特异性凋亡反应的情况下被减少/消除。这种siRNA/dsRNA方法将为检测精子发生过程中表达的蛋白质的功能提供一种非常有效的方法。这是一项相对简单的技术，需要建立一个转基因品系。此外，如果RNAi/dsRNA方法“敲低”但不“敲除”特定的蛋白质，则可能产生低形态表型，这将提供有关基因功能的额外信息，例如，蛋白质正常功能的阈值水平。

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.