Spatial and temporal ‘knock down’ of gene expression by electroporation of double-stranded RNA and morpholinos into early postimplantation mouse embryos

Abstract

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.

Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos

“…They produced a phenotype, although RNAi appeared to be a more robust approach in this case (Lefebvre et al, 2002). A slightly better RNAi effect compared to morpholinos has also been observed in early postimplantation mouse embryos (Mellitzer et al, 2002) but this higher efficiency of RNAi is not always the case, as observed for example in HeLa cells (Munshi et al, 2002). Therefore, differences in the mode of action, efficiency of delivery, toxicity, and cost may lead to different choices in different situations.…”

“…Importantly, Grabarek and coworkers developed protocols for electroporation of zonaenclosed embryos allowing studies involving embryo transfer of treated samples. Electroporation of pre-processed siRNA has been also successfully used in early postimplantation embryos (Mellitzer et al, 2002) Finally, it should be noted that experiments in the oocyte showed decreased RNAi efficiency when two genes were targeted simultaneously, suggesting that the RNAi pathway can be readily saturated so that simultaneous targeting of several genes may be difficult to achieve at this stage (Stein and Svoboda, 2003).…”

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

“…1B) (Mellitzer et al, 2002;Soares et al, 2008). However, it can be inferred from computations of the electric potential at the surface of an ellipsoid model that although permeation localisation is optimal when the longer axis of the embryo is perpendicular to both electrodes, it degrades as soon as a tilt angle exists, becoming nearly impossible after a 90°rotation (Valic et al, 2003).…”

“…A variety of molecules can be efficiently introduced into cells by electroporation, including DNA, mRNA (Cerda et al, 2006;Chernet and Levin, 2012), dsRNA (Mellitzer et al, 2002;Soares et al, 2008), morpholinos (Falk et al, 2007;Mellitzer et al, 2002;Voiculescu et al, 2008), siRNA (Calegari et al, 2004;Paganin-Gioanni et al, 2011), proteins and drugs (Teissie et al, 2012). In this work we concentrated on DNA electroporation to provide a readout for transfection.…”

Electroporation: an arsenal of application

“…At present many of the electroporations were used for testing the function of a gene with gain or loss strategies spatially and temporally using plasmid DNA, siRNA (Ghartey-Tagoe et al 2006), dsRNA and morpholinos (Mellitzer et al 2002) and so on. Ectopic expression was also performed to test the function of target protein (Xiang et al 2004).…”