Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo

Abstract

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

Figure 1

Representative image of co-localization of the TatU1A-Alexa546/RNA complex and LysoTracker green in a 2-cell stage embryo. We observed similar results in two more embryos. Scale bar, 30 μm.

Figure 2

PCDR-mediated shRNA delivery into single blastomere in embryos. Irradiated blastomeres are indicated by arrows in the light (+) images. RNA delivery in 2-cell stage (a), and 4-cell stage (b) embryos are shown. Scale bar, 30 μm.

Figure 3

EGFP knockdown in a single blastomere. (a) One blastomere was co-injected with EGFP and BFP mRNAs, and photoirradiated to deliver shGFP. (b) Two blastomeres were co-injected with EGFP and BFP mRNAs, and one blastomere was photoirradiated to internalize shGFP. (c) Two blastomeres were co-injected with EGFP and BFP mRNAs, and one blastomere was photoirradiated to internalize shCtrl. We confirmed photo-dependent EGFP knockdown in six other embryos. Scale bar, 30 μm.