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. ? ?
