Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals.We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.RNA interference (RNAi) is a sequence-specific mRNA degradation induced by double stranded RNA (dsRNA). Briefly, long dsRNA is processed in the cytoplasm by RNase III Dicer into 20 - 22 bp long short interfering RNAs (siRNAs), which are loaded on the effector RNA-induced silencing complex (RISC). siRNAs serve as guides for cleavage of complementary RNAs, which are cleaved in the middle of the duplex formed between a siRNA and its cognate RNA (reviewed in detail in [1]).RNAi is a widely used approach for inhibiting gene function in many eukaryotic model systems. Compared to other strategies for blocking gene functions, RNAi provides several advantages. It can be used to silence any gene, it is fast, relatively simple to use, and its cost is reasonably low. RNAi is usually induced either by delivering siRNAs or long dsRNAs into cells or by expressing RNA-inducing molecules from a vector. A number of strategies was developed for tissue-specific and inducible RNAi, thus offering an attractive alternative to traditional gene targeting by homologous recombination.RNAi became a favorable tool to block gene function also in mammalian oocytes. In fact, mouse oocytes were the first mammalian cell type where RNAi was used [2,3]. RNAi induced by microinjection of long dsRNA or siRNA into fully-grown germinal vesicle-intact (GV) oocytes is an excellent tool to study the