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

Abstract

BackgroundRNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type.ResultsHere 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.ConclusionsWe 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.

Pronuclear microinjection is not suitable for RNA polymerase III promoter driven constitutive RNAi transgenesis in mice for XY male-to-female sex reversal by <i>Sry</i> gene knockdown

“…The knockdown efficiency of the shRNA constructs in vivo may be confirmed by organ culture on agar blocks and magnetically induced transfection using embryonic genital ridges [14] or by injection into tail vein of pregnant mice [15]. For transgenic RNAi in mice, one-cell embryos [16][17][18][19][20][21][22][23][24][25][26][27][28][29] or embryonic stem (ES) cells [30][31][32][33][34][35][36][37] have been used as the target ( Table 1). The methods of ES cells need several months for many steps such as vector construction, clone isolation, chimera mouse production and breeding.…”

“…However ES cells have made possible a ubiquitously active locus (e.g., ColA1 and Rosa26) targeting transgene integration [34][35][36][37]. One-cell embryos have been used for pronuclear microinjection [16][17][18][19][20][21][22][23][24][25] or lentivirus infection [17,[26][27][28][29]. Long double strand RNA (dsRNA) (around 500bp) expressing vectors were constructed for microinjection [19][20][21][22][23].…”

“…Single copy insertion is attractive for RNAi transgenesis since it is expected that too high expression of shRNA should induce embryonic lethality in this study. Moreover high efficiency of transgenesis would make it easy to analyze F 0 Tg embryos and it should be useful tool when the [16][17][18] constitutive polII [19] oocyte specific polII [20][21][22][23] Cre/loxP [24] microinjection tetracycline-responsive polII [25] One-cell embryos lentiviral infection constitutive polIII [17,[26][27][28][29] electroporation constitutive polIII [30,31] constitutive polIII [28]…”

Using Engineered microRNAs as Vectors for Animal RNA Interference: Promises and Challenges

“…Many labs have utilized this technology to create shRNA libraries and stable transgenic lines [62] [65]. However, this strategy does not work in all organisms and in some systems suffers from low efficiency and high off-target effects [66]. Furthermore, since it appears that stable shRNA expression is only achievable using RNA polymerase III promoters, which are constitutively expressed at a certain level in all cell types, the ability to control the timing and level of shRNA expression is currently not possible [62].…”

shRNAPred (version 1.0): An open source and standalone software for short hairpin RNA (shRNA) prediction

“…shRNAs are pivotal in the field of gene silencing as these are cheaper than siRNAs for large-scale studies [2]. However, currently available web-based tools fail to predict shRNAs from a large set of nucleotide sequences.…”